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Abstract:

The invention relates to methods, reagents and devices for detection and
characterization of nucleic acids, cells, and other biological samples.
Assay method are provided in which a sample is partitioned into
sub-samples, and analysis of the contents of the sub-samples carried out.
The invention also provides microfluidic devices for conducting the
assay. The invention also provides an analysis method using a universal
primers and probes for amplification and detection.

Claims:

1. An assay method comprising (a) partitioning a sample into a plurality
of sub-samples, wherein said sample comprises a plurality of nucleic acid
molecules, and wherein at least two sub-samples comprise at least one
nucleic acid molecule; (b) providing sufficient reagents in each
sub-sample to amplify a target sequence or sequences; (c) amplifying the
target sequence(s) in the sub-sample(s) containing target sequence(s)
thereby producing amplicons in the sub-sample; (d) distributing the
amplicons into a plurality of aliquots; and, (e) for each aliquot,
determining a property of amplicons in the aliquot.

2. An assay method comprising (a) partitioning a sample into a plurality
of sub-samples, wherein said sample comprises a plurality of nucleic acid
molecules, and wherein at least two sub-samples comprise at least one
nucleic acid molecule; (b) providing sufficient reagents in each
sub-sample to amplify at least two different target sequences; (c)
amplifying target sequence(s) in at least two sub-sample(s) thereby
producing amplicons in the sub-sample(s); (d) combining the amplicons
from said at least two sub-samples to create an amplicon pool; (d)
dividing the amplicon pool into a plurality of aliquots; and, (e) for
each aliquot, determining a property of amplicons in the aliquot.

3. The method of claim 2 wherein the sample is partitioned into at least
10.sup.4 sub-samples.

4. The method of claim 3 wherein each subsample has a volume of less than
1 nanoliter.

6. The method of claim 2 wherein sufficient reagents are provided to
amplify at least 10, 20, or 50 different target sequences, if present.

7. The method of claim 2 wherein said amplification is by PCR or RT-PCR.

8. The method of claim 2 wherein the amplicon pool is divided into at
least 10, 20, 50 or 100 aliquiots.

9. The method of claim 2 wherein the sample comprises a plurality of
cells comprising nucleic acid molecules, and wherein partitioning the
sample comprises partitioning intact cells into a plurality of
sub-samples.

10. The method of claim 2 wherein the sample comprises only one cell.

11. A method for conducting an analysis, comprising: (a) partitioning a
sample comprising a plurality of separable cells into at least 1000
separate reaction chambers in a MPD, wherein after partitioning at least
two reaction chambers each comprise exactly one cell; (b) providing in
each reaction chamber one or more reagents for determining a property or
properties of a cell, wherein the same reagents are provided in each
chamber; and (c) determining at least two different properties of a
single cell in a chamber and/or determining at least one property for at
least two different cells in different chambers.

12. The method of claim 11 wherein at least 99% of the reaction chambers
contain zero or one cell.

15. The method of claim 11 wherein at least one property is the presence
or absence in the cell of a nucleic acid having a specified sequence.

16. The method of claim 11 wherein at least one property is other than
the presence or absence in the cell of a nucleic acid having a specified
sequence.

17. A method for amplification and detection of multiple target DNA
sequences in a sample, said method comprising a) providing a sample
containing i) multiple target DNA sequences, ii) a primer pair
corresponding to each of said multiple target DNA sequences, each pair
consisting of a first primer comprising U1, B1 and F domains in
the order 5'-U1-B1-F-3' and a second primer comprising U2
and R domains in the order 5'-U2-R-3', wherein each pair of F and R
primers is capable of annealing specifically to a different target DNA
sequence under stringent annealing conditions; iii) a universal primer
pair capable of amplifying a double stranded DNA molecule with the
structure 5'U1-----U2'-3' 3'U1'-----U2-3' where
U1' is the sequence complementary to U1 and U2' is the
sequence complementary to U2; b) subjecting the sample to multiple
cycles of melting, reannealing, and DNA synthesis thereby producing
amplicons for each of said multiple target DNA sequences, and c)
detecting the amplicons using a probe that anneals to sequence of the
amplicon having the sequence of the B1 domain or its complement.

18. The method of claim 17 wherein the sample also contains a second set
of multiple target sequences, a primer pair corresponding to to each of
the target sequences in the second set, each pair consisting of a first
primer comprising U1, B2 and F domains in the order
5'-U1-B2-F-3' and a second primer comprising U2 and R
domains in the order 5'-U2-R-3', wherein each pair of F and R
oligonucleotides is capable of annealing specifically to a different
target DNA sequence in the second set of multiple target sequences under
stringent annealing conditions; and wherein amplicons for each of the
multiple target DNA sequences of the second set are produced; and
detecting the amplicons for each of the multiple target DNA sequences
using a probe that anneals to sequence of the amplicon having the
sequence of the B2 domain or its complement.

19. The method of claim 17 wherein U1, B1, F1, U2 and
R1 domains are between 6 and 25 nucleotides in length and the probe
is a molecular beacon.

20. A microfluidic device, comprising (a) a first region comprising (i) a
flow channel formed within an elastomeric material and having a first end
and a second end in fluid communication with each other through said
channel, wherein said channel may be branched or unbranched; (ii) an
inlet for introducing a sample fluid in communication with said channel,
said inlet; (iii) an outlet in communication with said flow channel; (iv)
a plurality of control channels overlaying the flow channel(s), wherein
an elastomeric membrane separates the control channels from the flow
channels at each intersection, the elastomeric membrane disposed to be
deflected into or withdrawn from the flow channel in response to an
actuation force, and wherein, when the control channels are actuated the
flow channel is partitioned into at least 1000 reaction chambers not in
fluidic communication with each other; (b) a second region compromising a
channel or chamber interposed between and in communication with said
outlet in (a) and a flow channel in the third region; (c) a third region
comprising a plurality of flow channels in fluidic communication with the
channel or chamber of the second region, with a region of each flow
channel defining a reaction site; (d) a control channel or channels that
when actuated separates the first and second regions; (e) a control
channel or channels that when actuated separates the second and third
regions; and (f) a control channel or channels that when actuated
separates the reaction sites of said flow channels from the other
portions of control channels.

21. The device of claim 20 wherein the flow channels in the third region
are blind flow channels.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional application No.
60/687,010, filed Jun. 2, 2005, the entire contents of which are herein
incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates to methods, reagents and devices for
detecting and characterizing nucleic acids, cells, and other biological
samples.

BACKGROUND

[0003] A variety of nucleic acid amplification assays and immunological
assays are used for analysis of cells and nucleic acids. These assays can
be used to detect or characterize nucleic acid sequences associated with
particular diseases or genetic disorders, for genotyping, for gene
expression analyses, to detect and identify pathogens such as viruses,
bacteria and fungi), for paternity and forensic identification, and for
many other purposes. However, in some applications the efficiency and
sensitivity of these assays is reduced, which may render the assays
useless or at minimum require that additional manipulations and/or
significant amounts of expensive reagents be used. For example, when a
cell or molecule to be analyzed is from a sample with a large excess of
non-target cells or molecules (e.g., as in genetic or phenotypic analysis
of a rare cell in a background of other cells) conventional assay methods
are inadequate. Similarly, when a number of different targets must be
detected in a single sample, conventional approaches (e.g., multiplex
PCR) are expensive, inefficient or not sufficiently sensitive. Thus, new
methods, reagents and devices for detection and characterization of
nucleic acids, cells, and other biological molecules will find broad
application in biomedicine and other fields.

BRIEF SUMMARY

[0004] The invention relates to methods, reagents and devices for
detection and characterization of nucleic acids, cells, and other
biological samples. In one aspect, the invention provides an assay method
including the following steps (a) partitioning a sample into a plurality
of sub-samples, where said sample comprises a plurality of nucleic acid
molecules, and where at least two sub-samples comprise at least one
nucleic acid molecule; (b) providing sufficient reagents in each
sub-sample to amplify a target sequence or sequences; (c) amplifying the
target sequence(s) in the sub-sample(s) containing target sequence(s)
thereby producing amplicons in the sub-sample; (d) distributing the
amplicons into a plurality of aliquots; and, (e) for each aliquot,
determining a property of amplicons in the aliquot.

[0005] In a related aspect, the invention provides an assay method
including the following steps (a) partitioning a sample into a plurality
of sub-samples, where said sample comprises a plurality of nucleic acid
molecules, and where at least two sub-samples comprise at least one
nucleic acid molecule; (b) providing sufficient reagents in each
sub-sample to amplify at least two different target sequences; (c)
amplifying target sequence(s) in at least two sub-sample(s) thereby
producing amplicons in the sub-sample(s); (d) combining the amplicons
from said at least two sub-samples to create an amplicon pool; (d)
dividing the amplicon pool into a plurality of aliquots; and, (e) for
each aliquot, determining a property of amplicons in the aliquot. In one
embodiment, the sample is partitioned into at least 104 subsamples.
In one embodiment, each subsample has a volume of less than one
nanoliter. In one embodiment, the nucleic acid molecules comprise DNA
and/or mRNA. In one embodiment, the amplification is by PCR or RT-PCR. In
one embodiment, sufficient reagents are provided to amplify at least 10,
20, or 50 different target sequences, if present. In one embodiment, the
amplicon pool is divided into at least 10, 20, 50 or 100 aliquiots. In
one embodiment, the sample contains a plurality of cells having nucleic
acid molecules, and partitioning the sample involves partitioning intact
cells into a plurality of sub-samples. In one embodiment, the sample
contains only one cell.

[0006] In another aspect, the invention provides an assay method including
the following steps (a) partitioning a sample comprising a plurality of
separable cells into at least 1000 separate reaction chambers in a
massively partitioning device (MPD), where after partitioning at least
two reaction chambers each comprise exactly one cell; (b) providing in
each reaction chamber one or more reagents for determining a property or
properties of a cell, where the same reagents are provided in each
chamber; and (c) determining at least two different properties of a
single cell in a chamber and/or determining at least one property for at
least two different cells in different chambers. In one embodiment, at
least 99% of the reaction chambers contain zero or one cell. In one
embodiment, the cells are bacterial cells. In one embodiment, the
reagents include reagents for nucleic acid amplification. In one
embodiment, at least one property is the presence or absence in the cell
of a nucleic acid having a specified sequence. In one embodiment, at
least one property is other than the presence or absence in the cell of a
nucleic acid having a specified sequence.

[0007] In another aspect, the invention provides a method for
amplification and detection of multiple target DNA sequences in a sample,
including the following steps: (a) providing a sample containing (i)
multiple target DNA sequences, (ii) a primer pair corresponding to each
of said multiple target DNA sequences, each pair consisting of a first
primer comprising U1, B1 and F domains in the order
5'-U1-B1-F-3' and a second primer comprising U2 and R
domains in the order 5'-U2-R-3', where each pair of F and R primers
is capable of annealing specifically to a different target DNA sequence
under stringent annealing conditions; (iii) a universal primer pair
capable of amplifying a double stranded DNA molecule with the structure

5'-U1--U2-3'

3-U1'---U2-3'

where U1' is the sequence complementary to U1 and U2' is
the sequence complementary to U2; (b) subjecting the sample to
multiple cycles of melting, reannealing, and DNA synthesis thereby
producing amplicons for each of said multiple target DNA sequences, and
(c) detecting the amplicons using a probe that anneals to sequence of the
amplicon having the sequence of the B1 domain or its complement. In
one embodiment, the sample also contains a second set of multiple target
sequences, a primer pair corresponding to to each of the target sequences
in the second set, each pair consisting of a first primer comprising
U1, B2 and F domains in the order 5'-U1-B2-F-3' and a
second primer comprising U2 and R domains in the order
5'-U2-R-3', where each pair of F and R oligonucleotides is capable
of annealing specifically to a different target DNA sequence in the
second set of multiple target sequences under stringent annealing
conditions; and where amplicons for each of the multiple target DNA
sequences of the second set are produced; and detecting the amplicons for
each of the multiple target DNA sequences using a probe that anneals to
sequence of the amplicon having the sequence of the B2 domain or its
complement. In one embodiment, U1, B1, F1, U2 and
R1 domains are between 6 and 30 nucleotides in length. In one
embodiment the probe is a molecular beacon. In one embodiment, the probe
is a a Taqman®-type probe.

[0008] In another aspect, the invention provides a microfluidic device,
having (a) a first region comprising (i) a flow channel formed within an
elastomeric material and having a first end and a second end in fluid
communication with each other through said channel, where said channel
may be branched or unbranched; (ii) an inlet for introducing a sample
fluid in communication with said channel, said inlet; (iii) an outlet in
communication with said flow channel; (iv) a plurality of control
channels overlaying the flow channel(s), where an elastomeric membrane
separates the control channels from the flow channels at each
intersection, the elastomeric membrane disposed to be deflected into or
withdrawn from the flow channel in response to an actuation force, and
where, when the control channels are actuated the flow channel is
partitioned into at least 1000 reaction chambers not in fluidic
communication with each other; (b) a second region compromising a channel
or chamber interposed between and in communication with said outlet in
(a) and a flow channel in the third region; (c) a third region comprising
a plurality of flow channels (e.g., blind flow channels), in fluidic
communication with the channel or chamber of the second region, with a
region of each flow channel defining a reaction site; (d) a control
channel or channels that when actuated separates the first and second
regions; (e) a control channel or channels that when actuated separates
the second and third regions; and (f) a control channel or channels that
when actuated separates the reaction sites of said flow channels from the
other portions of control channels.

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIGS. 1A and 1B show an exemplary design of a massively
partitioning device (MPD) in valve off (FIG. 1A) and valve actuated (FIG.
1B) states.

[0010]FIG. 2 shows an exemplary design of a MPD with two banks: a first
bank in which nucleic acids are partitioned and amplified in individual
chambers, and a second bank in which subsequent analysis of the amplicon
pool occurs.

[0011] FIGS. 3A-C are flow charts illustrating partition and analysis of
nucleic acids using methods of the invention. FIG. 3A illustrates
partition and analysis of nucleic acids in which multiple target
sequences are amplified. FIG. 3B illustrates partition and analysis acids
in which target sequences in only a single chamber are amplified. FIG. 3c
illustrates analysis of nucleic acids of a single cell.

[0012]FIG. 4 is a flow chart illustrating partition of cells and analysis
of their properties.

[0013]FIG. 5 is an illustration of primers used in the universal
amplification method.

DETAILED DESCRIPTION

Definitions

[0014] The term "elastomer" has the general meaning used in the art. Thus,
for example, Allcock et al. (Contemporary Polymer Chemistry, 2nd Ed.)
describes elastomers in general as polymers existing at a temperature
between their glass transition temperature and liquefaction temperature.
Elastomeric materials exhibit elastic properties because the polymer
chains readily undergo torsional motion to permit uncoiling of the
backbone chains in response to a force, with the backbone chains
recoiling to assume the prior shape in the absence of the force. In
general, elastomers deform when force is applied, but then return to
their original shape when the force is removed. The elasticity exhibited
by elastomeric materials can be characterized by a Young's modulus. The
elastomeric materials utilized in the microfluidic devices disclosed
herein typically have a Young's modulus of between about 1 Pa-1 TPa, in
other instances between about 10 Pa-100 GPa, in still other instances
between about 20 Pa-1 GPa, in yet other instances between about 50 Pa-10
MPa, and in certain instances between about 100 Pa-1 MPa. Elastomeric
materials having a Young's modulus outside of these ranges can also be
utilized depending upon the needs of a particular application.
Microfluidic devices can be fabricated from an elastomeric polymer such
as GE RTV 615 (formulation), a vinyl-silane crosslinked (type) silicone
elastomer (family). However, elastomeric microfluidic systems are not
limited to this one formulation, type or even this family of polymer;
rather, nearly any elastomeric polymer is suitable. Given the tremendous
diversity of polymer chemistries, precursors, synthetic methods, reaction
conditions, and potential additives, there are a large number of possible
elastomer systems that can be used to make monolithic elastomeric
microvalves and pumps (including, for example, perfluoropolyethers,
polyisoprene, polybutadiene, polychloroprene, polyisobutylene,
poly(styrene-butadiene-styrene), polyurethanes, and silicones, for
example, or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),
poly(carborane-siloxanes) (Dexsil), polyacrylonitrile-butadiene) (nitrile
rubber), poly(1-butene), poly(chlorotrifluoroethylene-vinylidene
fluoride) copolymers (Kel-F), poly(ethyl vinyl ether), poly(vinylidene
fluoride), poly(vinylidene fluoride-hexafluoropropylene) copolymer
(Viton), elastomeric compositions of polyvinylchloride (PVC),
polysulfone, polycarbonate, polymethylmethacrylate (PMMA),
polytertrafluoroethylene (Teflon), polydimethylsiloxane,
polydimethylsiloxane copolymer, and aliphatic urethane diacrylate). The
choice of materials typically depends upon the particular material
properties (e.g., solvent resistance, stiffness, gas permeability, and/or
temperature stability) required for the application being conducted.
Additional details regarding the type of elastomeric materials that can
be used in the manufacture of the components of the microfluidic devices
disclosed herein are set forth in Unger et al. (2000) Science
288:113-116, PCT Publications WO 02/43615, WO 2005030822, WO 2005084191
and WO 01/01025; and U.S. patent publication No. 20050072946.

[0015] A "reagent" refers broadly to any agent used in a reaction, other
than the analyte (e.g., cell or nucleic acid being analyzed). Exemplary
reagents for a nucleic acid amplification reaction include, but are not
limited to, buffer, metal ions, polymerase, reverse transcriptase,
primers, template nucleic acid, nucleotides, labels, dyes, nucleases and
the like. Reagents for enzyme reactions include, for example, substrates,
cofactors, buffer, metal ions, inhibitors and activators. Reagents for
cell-based reactions include, but are not limited to, cells, cell
specific dyes and ligands (e.g., agonists and antagonists) that bind to
cellular receptors.

[0016] The terms "nucleic acid," "polynucleotide," and "oligonucleotide"
include a polymeric form of nucleotides of any length, including, but not
limited to, ribonucleotides or deoxyribonucleotides. There is no intended
distinction in length between these terms. Further, these terms refer
only to the primary structure of the molecule. Thus, in certain
embodiments these terms can include triple-, double- and single-stranded
DNA, as well as triple-, double- and single-stranded RNA. They also
include modifications, such as by methylation and/or by capping, and
unmodified forms of the polynucleotide. More particularly, the terms
"nucleic acid," "polynucleotide," and "oligonucleotide," include
polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
polyribonucleotides (containing D-ribose), any other type of
polynucleotide which is an N- or C-glycoside of a purine or pyrimidine
base, and other polymers containing nonnucleotidic backbones, for
example, polyamide (e.g., peptide nucleic acids (PNAs)) and
polymorpholino (commercially available from the Anti-Virals, Inc.,
Corvallis, Oreg., as Neugene) polymers, and other synthetic
sequence-specific nucleic acid polymers providing that the polymers
contain nucleobases in a configuration which allows for base pairing and
base stacking, such as is found in DNA and RNA.

[0017] A "primer" is a single-stranded polynucleotide capable of acting as
a point of initiation of template-directed DNA or RNA synthesis under
appropriate conditions (i.e., in the presence of four different
nucleoside triphosphates and an agent for polymerization, such as, DNA or
RNA polymerase or reverse transcriptase) in an appropriate buffer and at
a suitable temperature. The appropriate length of a primer depends on the
intended use of the primer but typically is at least 7 nucleotides long
and, more typically range from 10 to 30 nucleotides in length. Other
primers can be somewhat longer such as 30 to 50 nucleotides long. In this
context, primer "length" refers to the portion of an oligo- or
polynucleotide that hybridizes to a complementary "target" sequence and
primes synthesis. For example, in the primer 5'-U1-B1-F1-3' the
length of F1 might be 20 nucleotides and the combined length of U, B
and F1 could be 60 nucleotides or more (typically between 30 and 100
nucleotides). Short primer molecules generally require cooler
temperatures to form sufficiently stable hybrid complexes with the
template. A primer need not reflect the exact sequence of the template
but must be sufficiently complementary to hybridize with a template. The
term "primer site" or "primer binding site" refers to the segment of the
target DNA to which a primer hybridizes. The term "primer pair" means a
set of primers including a 5' "upstream primer" that hybridizes with the
complement of the 5' end of the DNA sequence to be amplified and a 3'
"downstream primer" that hybridizes with the 3' end of the sequence to be
amplified.

[0018] A primer or probe anneals or hybridizes to a complementary target
sequence. The primer or probe may be exactly complementary to the target
sequence or can be less than perfectly complementary. Typically the
primer has at least 65% identity to the complement of the target sequence
over a region of at least 7 nucleotides, more typically over a region in
the range of 10-30 nucleotides, and often over a region of at least 14-25
nucleotides, and more often has at least 75% identity, at least 85%
identity or 90% identity. It will be understood that certain bases (e.g.,
the 3' base of a primer) generally should be exactly complementary to
corresponding base of the target sequence. Primer and probes generally
anneal to the target sequence under stringent conditions. Stringent
annealing conditions refers to conditions in a range from about 5°
C. to about 20° C. or 25° C. below the melting temperature
(Tm) of the target sequence and a probe with exact or nearly exact
complementarity to the target. As used herein, the melting temperature is
the temperature at which a population of double-stranded nucleic acid
molecules becomes half-dissociated into single strands. Methods for
calculating the Tm of nucleic acids are well known in the art (see,
e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO
MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and
Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED.,
VOLS. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by
reference). As indicated by standard references, a simple estimate of the
Tm value may be calculated by the equation: Tm=81.5+0.41(%
G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g.,
Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID
HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the
conditions for stringent hybridization) is affected by various factors
such as the length and nature (DNA, RNA, base composition) of the probe
and nature of the target (DNA, RNA, base composition, present in solution
or immobilized, and the like), and the concentration of salts and other
components (e.g., the presence or absence of formamide, dextran sulfate,
polyethylene glycol). The effects of these factors are well known and are
discussed in standard references in the art

[0019] A "probe" is an nucleic acid capable of binding to a target nucleic
acid of complementary sequence through one or more types of chemical
bonds, usually through complementary base pairing, usually through
hydrogen bond formation, thus forming a duplex structure. The probe binds
or hybridizes to a "probe binding site." The probe can be labeled with a
detectable label to permit facile detection of the probe, particularly
once the probe has hybridized to its complementary target. The label
attached to the probe can include any of a variety of different labels
known in the art that can be detected by chemical or physical means, for
example. Suitable labels that can be attached to probes include, but are
not limited to, radioisotopes, fluorophores, chromophores, mass labels,
electron dense particles, magnetic particles, spin labels, molecules that
emit chemiluminescence, electrochemically active molecules, enzymes,
cofactors, and enzyme substrates. Probes can vary significantly in size.
Some probes are relatively short. Generally, probes are at least 7 to 15
nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides
long. Still other probes are somewhat longer, being at least 50, 60, 70,
80, 90 nucleotides long. Yet other probes are longer still, and are at
least 100, 150, 200 or more nucleotides long. Probes can be of any
specific length that falls within the foregoing ranges as well.

[0020] The term "label" refers to a molecule or an aspect of a molecule
that can be detected by physical, chemical, electromagnetic and other
related analytical techniques. Examples of detectable labels that can be
utilized include, but are not limited to, radioisotopes, fluorophores,
chromophores, mass labels, electron dense particles, magnetic particles,
spin labels, molecules that emit chemiluminescence, electrochemically
active molecules, enzymes, cofactors, enzymes linked to nucleic acid
probes and enzyme substrates. The term "detectably labeled" means that an
agent has been conjugated with a label or that an agent has some inherent
characteristic (e.g., size, shape or color) that allows it to be detected
without having to be conjugated to a separate label.

[0021] A "polymorphic marker" or "polymorphic site" is the locus at which
divergence occurs. Preferred markers have at least two alleles, each
occurring at frequency of greater than 1%, and more preferably greater
than 10% or 20% of a selected population. A polymorphic locus may be as
small as one base pair. Polymorphic markers include restriction fragment
length polymorphisms, variable number of tandem repeats (VNTR's),
hypervariable regions, minisatellites, dinucleotide repeats,
trinucleotide repeats, tetranucleotide repeats, simple sequence repeats,
and insertion elements such as Alu. The first identified allelic form is
arbitrarily designated as the reference form and other allelic forms are
designated as alternative or variant alleles. The allelic form occurring
most frequently in a selected population is sometimes referred to as the
wildtype form. Diploid organisms may be homozygous or heterozygous for
allelic forms. A diallelic polymorphism has two forms. A triallelic
polymorphism has three forms.

[0022] A "single nucleotide polymorphism" (SNP) occurs at a polymorphic
site occupied by a single nucleotide, which is the site of variation
between allelic sequences. The site is usually preceded by and followed
by highly conserved sequences of the allele (e.g., sequences that vary in
less than 1/100 or 1/1000 members of the populations). A single
nucleotide polymorphism usually arises due to substitution of one
nucleotide for another at the polymorphic site. A transition is the
replacement of one purine by another purine or one pyrimidine by another
pyrimidine. A transversion is the replacement of a purine by a pyrimidine
or vice versa. Single nucleotide polymorphisms can also arise from a
deletion of a nucleotide or an insertion of a nucleotide relative to a
reference allele.

[0023] The term "haplotype" refers to the designation of a set of
polymorphisms or alleles of polymorphic sites within a gene of an
individual.

[0024] A used herein, "plurality" means at least three. In general a
plurality of cells, nucleic acid molecules, etc., will contain at least
10, at least about 102, at least about 103, or at least about
104 different cells, molecules, etc.

[0025] A used herein, "entities" refers to a plurality of structurally
similar biological molecules or structures (e.g., macromolecules such as
nucleic acids, protein, carbohydrates and lipids; cells or subcellular
structures or components; viruses) or nonbiological particles that are
separate and distinct from each other in the sense that they can be
separated into separate reaction chambers using a MPD. "Entity" refers to
a single such molecule or structure.

[0026] The term "biological sample", refers to a sample obtained from an
organism or from components of an organism, such as cells, biological
tissues and fluids. In some methods, the sample is from a human patient.
Such samples include sputum, blood, blood cells (e.g., white cells),
tissue or fine needle biopsy samples, urine, peritoneal fluid, and
fleural fluid, or cells therefrom.

A. Introduction

[0027] The invention relates generally to analysis of macromolecules and
small particles, and particularly to analysis of nucleic acids, proteins,
and individual cells. In certain aspects, the invention relates to
analysis methods involving massive partitioning. Massive partitioning of
liquid samples, i.e., dividing the sample into thousands of isolated
reaction volumes, has been made possible by the development of specially
designed elastomeric microfluidic devices. These devices can be referred
to as "massively partitioning devices" or MPDs and are sometimes referred
to as "chips" or Digital Isolation and Detection Integrated Fluidic
Circuits (DID IFCs). Exemplary devices are described in McBride et al.
(PCT publication WO 2004/089,810, published on Oct. 21, 2004; copending,
commonly assigned U.S. patent application Ser. No. 10/819,088 published
as patent publication No. 20050019792 on Jan. 27, 2005; and copending,
commonly assigned U.S. patent application Ser. No. 10/819,088 published
as patent publication No. 20050252773 on Nov. 17, 2005, each of which is
incorporated by reference in its entirety for all purposes and the
specific purposes describe therein and herein; hereinafter referred to
together as "McBride et al."). Using MPDs, a sample can be partitioned
into a multitude of isolated reaction chambers, and reactions carried out
simultaneously in each chamber. For example, McBride et al., supra,
describes the performance of 21,000 simultaneous PCR reactions in a
single microfluidic chip, in a volume of 90 picoliters per reaction and
with single template molecule sensitivity.

[0028] In a first broad aspect, the invention provides new methods and
devices for analysis of a sample containing nucleic acids, proteins,
other biomolecules, cells, microorganisms, viruses, and other biological
or nonbiological entities, in which the sample undergoes massive
partitioning as part of the analysis process.

[0029] In a second broad aspect, the invention provides methods and
reagents for amplification and/or detection of a nucleic acid. These
methods and reagents find particular application in the analysis of
nucleic acids partitioned using a MPD, but may be used in
amplification-based analysis of any nucleic acid.

[0030] These and other inventions are described in the following sections.

B. Massively Partitioning Devices

[0031] Methods described in this disclosure can be, and in some cases are
necessarily, carried out using an elastomeric microfluidic device.
Methods for fabricating elastomeric microfluidic devices capable of
separating molecules or cells and for carrying out reactions are known in
the art (see, e.g., Unger et al., 2000, Science 288:113-116, PCT
Publications WO 01/01025 and WO/02/43615; and U.S. patent application
Ser. No. 10/306,798 published as Pat App. No. 20030138829 on Jul. 24,
2003). In particular, exemplary elastomeric massively partitioning
devices (MPDs) are described in McBride et al., supra and references
cited therein. Based on these and other publications, one of ordinary
skill in the art guided by this disclosure will be able to practice all
aspects of the inventions described herein. Accordingly, elastomeric
microfluidic devices are described only briefly below.

General Structure of Microfluidic Devices

[0032] Elastomeric microfluidic devices are characterized in part by
utilizing various components such as flow channels, control channels,
valves, pumps, vias, and/or guard channels from elastomeric materials.
FIGS. 1A and 1B show an exemplary design of a massively partitioning
device.

[0033] A "flow channel" refers generally to a flow path through which a
solution can flow. A "blind channel" refers to a flow channel which has
an entrance but not a separate exit. A "control channel" is a channel
separated from a flow channel by an elastomeric membrane that can be
deflected into or retracted from the flow channel in response to an
actuation force. The term "valve" refers to a configuration in which a
flow channel and a control channel intersect and are separated by an
elastomeric membrane that can be deflected into or retracted from the
flow channel in response to an actuation force. An "isolated reaction
site" or "reaction chamber" refers to a reaction site that is not in
fluid communication with other reactions sites present on the device, and
which is created by the actuation of control channels in the device. A
"via" refers to a channel formed in an elastomeric device to provide
fluid access between an external port of the device and one or more flow
channels. Thus, a via can serve as a sample input or output, for example.
"Guard channels" may be included in elastomeric microfluidic devices for
use in heating applications to minimize evaporation of sample from the
reaction sites. Guard channels are channels formed within the elastomeric
device through which water can be flowed, to increase the water vapor
pressure within the elastomeric material from which the device is formed,
thereby reducing evaporation of sample from the reaction sites. The guard
channels are similar to the control channels in that typically they are
formed in a layer of elastomer that overlays the flow channels and/or
reaction site. Typically, the guard channels are placed adjacent and over
flow channels and reaction sites as these are the primary locations at
which evaporation is the primary concern. Guard channels are typically
formed in the elastomer utilizing the MSL techniques and/or
sacrificial-layer encapsulation methods cited above. The solution flowed
through the guard channel includes any substance that can reduce
evaporation of water.

[0034] The devices incorporate flow channels, control channels and valves
to isolate selectively a reaction site at which reagents are allowed to
react. FIGS. 1A and 1B depict an exemplary design of a partitioning
microfluidic device 01 in a valve off and valve actuated state. Referring
to the figure, a sample is injected into inlet 02 which is in
communication with branched partitioning channel system 03 of the device.
Solution flow through flow channels of the device is controlled, at least
in part, with one or more control channels that are separated from the
flow channel by an elastomeric membrane or segment. This membrane or
segment can be deflected into or retracted from the flow channel with
which a control channel is associated by applying an actuation force to
the control channels so that solution flow can be entirely blocked by
valves. Actuating the control valves creates isolated reaction chambers
05 in which individual reactions can be conducted. The reaction chambers
can number from 103 to 105 or more be at a density of at least
100 sites/cm2 and can range up to at least 2000, 3000, 4000 or more
than 4000 sites/cm2. Very small wells or cavities can be formed
within an elastomeric material to increase the volume of the reaction
chamber. Valves can be actuated by injecting gases (e.g., air, nitrogen,
and argon), liquids (e.g., water, silicon oils and other oils), solutions
containing salts and/or polymers (including but not limited to
polyethylene glycol, glycerol and carbohydrates), and the like into the
port.

[0035] Although FIG. 1 illustrates a MPD with branched flow channels, any
channel configuration (flow channel path) that can be partitioned by
control channels can be used in accordance with the invention, including,
for example, square, spiral or serpentine configurations.

[0036] The dimensions of flow channels in a MPD can vary widely. Typically
channels are from about 0.1 μm to about 1000 μm in any dimension,
sometimes from about 0.1 to about 100 μm, and sometimes from about 0.1
to about 10 μm. In one embodiment the channels have a high aspect
ratio (e.g., a height to width ratio of from about 2:1 to about 10:1) to
increase channel density and/or to increase signal collection from
channels containing a detectably labeled moiety. For example, in some
embodiments the channel has a columnar shape in which the dimensions of
floor and ceiling are smaller that the dimensions of the walls, and a
signal (e.g., fluorescence, infra red or visible radiation) is detected
through the ceiling or floor. Appropriate channel dimensions will depend
in part on the nature of the entities being partitioned. For partition of
eukaryotic cells, for example, a dimension should be at least sufficient
for passage of the cell (e.g., 2-5 times the dimension of the cell).
However, for the purpose of restricting movement the dimensions can be on
the order of 0.75 times the smallest dimension of the particle.
Microfluidic manipulation and analysis of particles is also described in
U.S. Patent Pub. 20040229349 entitled "Microfluidic particle-analysis
systems" and incorporated herein by reference.

[0037] Reactions (e.g., nucleic acid amplification, protein binding, etc.)
are allowed to occur in each chamber. For example, PCR reactions can be
initiated by heating the chambers (e.g., placing the device on a suitably
programmed flat plate thermocycler.

[0038] The results or products of the reaction can be detected using any
of a number of different detection strategies. Because the MPDs are
usually made of elastomeric materials that are relatively optically
transparent, reactions can be readily monitored using a variety of
different detection systems at essentially any location on the
microfluidic device. Most typically, however, detection occurs at the
reaction site itself.

[0039] The nature of the signal to be detected will, of course, determine,
to a large extent, the type of detector to be used. The detectors can be
designed to detect a number of different signal types including, but not
limited to, signals from radioisotopes, fluorophores, chromophores,
electron dense particles, magnetic particles, spin labels, molecules that
emit chemiluminescence, electrochemically active molecules, enzymes,
cofactors, enzymes linked to nucleic acid probes and enzyme substrates.
Illustrative detection methodologies suitable for use with the present
microfluidic devices include, but are not limited to, light scattering,
multichannel fluorescence detection, infra-red, UV and visible wavelength
absorption, luminescence, differential reflectivity, and confocal laser
scanning. Additional detection methods that can be used in certain
application include scintillation proximity assay techniques,
radiochemical detection, fluorescence polarization, fluorescence
correlation spectroscopy (FCS), time-resolved energy transfer (TRET),
fluorescence resonance energy transfer (FRET) and variations such as
bioluminescence resonance energy transfer (BRET). Additional detection
options include electrical resistance, resistivity, impedance, and
voltage sensing.

[0040] A detector can include a light source for stimulating a reporter
that generates a detectable signal. The type of light source utilized
depends in part on the nature of the reporter being activated. Suitable
light sources include, but are not limited to, lasers, laser diodes and
high intensity lamps. If a laser is utilized, the laser can be utilized
to scan across a set of detection sections or a single detection section.
Laser diodes can be microfabricated into the microfluidic device itself.
Alternatively, laser diodes can be fabricated into another device that is
placed adjacent to the microfluidic device being utilized to conduct a
thermal cycling reaction such that the laser light from the diode is
directed into the detection section.

[0041] Detectors can be microfabricated within the microfluidic device, or
can be a separate element. A number of commercially-available external
detectors can be utilized. Many of these are fluorescent detectors
because of the ease in preparing fluorescently labeled reagents. Specific
examples of detectors that are available include, but are not limited to,
Applied Precision ArrayWoRx (Applied Precision, Issaquah, Wash.) and the
ABI 7700 (Applied Biosystems, Inc., Foster City, Calif.).

Fabrication

[0042] Microfluidic devices are generally constructed utilizing single and
multilayer soft lithography (MSL) techniques and/or sacrificial-layer
encapsulation methods. The basic MSL approach involves casting a series
of elastomeric layers on a micro-machined mold, removing the layers from
the mold and then fusing the layers together. In the sacrificial-layer
encapsulation approach, patterns of photoresist are deposited wherever a
channel is desired. These techniques and their use in producing
microfluidic devices is discussed in detail, for example, by Unger et
al., 2000, Science 288:113-116; by Chou, et al., 2000, "Integrated
Elastomer Fluidic Lab-on-a-chip-Surface Patterning and DNA Diagnostics,
in Proceedings of the Solid State Actuator and Sensor Workshop, Hilton
Head, S.C.; in PCT Publication WO 01/01025; and in published U.S. patent
application No. 20050072946 (each incorporated herein by reference).

[0043] In one approach, the foregoing fabrication methods initially
involve fabricating mother molds for top layers (e.g., the elastomeric
layer with the control channels) and bottom layers (e.g., the elastomeric
layer with the flow channels) on silicon wafers by photolithography with
photoresist (Shipley SJR 5740). Channel heights can be controlled
precisely by the spin coating rate. Photoresist channels are formed by
exposing the photoresist to UV light followed by development. Heat reflow
process and protection treatment is typically achieved as described by
Unger et al. supra. A mixed two-part-silicone elastomer (GE RTV 615) is
then spun into the bottom mold and poured onto the top mold,
respectively. Spin coating can be utilized to control the thickness of
bottom polymeric fluid layer. The partially cured top layer is peeled off
from its mold after baking in the oven at 80° C. for 25 minutes,
aligned and assembled with the bottom layer. A 1.5-hour final bake at
80° C. is used to bind these two layers irreversibly. Once peeled
off from the bottom silicon mother mold, this RTV device is typically
treated with HCL (0.1N, 30 min at 80° C.). This treatment acts to
cleave some of the Si--O--Si bonds, thereby exposing hydroxy groups that
make the channels more hydrophilic.

[0044] The device can then optionally be hermetically sealed to a support.
The support can be manufactured of essentially any material, although the
surface should be flat to ensure a good seal, as the seal formed is
primarily due to adhesive forces. Examples of suitable supports include
glass, plastics and the like.

[0045] In certain devices, the devices formed according to the foregoing
method result in the substrate (e.g., glass slide) forming one wall of
the flow channel. Alternatively, the device once removed from the mother
mold is sealed to a thin elastomeric membrane such that the flow channel
is totally enclosed in elastomeric material. For certain uses, e.g., PCR
amplification, flow channels and chambers enclosed in elastomeric
material (i.e., without a glass wall) are preferred. The resulting
elastomeric device can then optionally be joined to a substrate support.
In some cases, the device is made as described in U.S. patent publication
No. 20050072946. In some cases, the device uses "push-up valves"
described in U.S. patent publication No. 20050072946 (e.g., FIG. 37B).
"Push-up" refers to low actuation pressure geometry in which the membrane
deflects upwards to seal off the upper fluid channel. In this geometry,
the deflectable membrane is featureless and exhibits a substantially
constant thickness.

[0046] Reagents can be deposited in reaction chambers before addition of a
sample to the MPD. A number of commercially available reagent spotters
and established spotting techniques can be used to deposit the
reagent(s). Microfluidic devices in which reagents are deposited at the
reaction sites during manufacture are typically formed of three layers.
The bottom layer is the layer upon which reagents are deposited. The
bottom layer can be formed from various elastomeric materials as
described in the references cited above on MLS methods. Typically, the
material is polydimethylsiloxane (PDMS) elastomer. Based upon the
arrangement and location of the reaction sites that is desired for the
particular device, one can determine the locations on the bottom layer at
which the appropriate reagents should be spotted. Because PDMS is
hydrophobic, the deposited aqueous spot shrinks to form a very small
spot. The deposited reagents are deposited such that a covalent bond is
not formed between the reagent and the surface of the elastomer because,
as described earlier, the reagents are intended to dissolve in the sample
solution once it is introduced into the reaction site. In some versions,
the reagent is designed to be inactive or unavailable to a reaction until
a specified condition occurs (e.g., a polymerase not activated until
heated or until the addition of a necessary cofactor).

[0047] The other two layers of the device are the layer in which the flow
channels are formed and the layer in which the control and optionally
guard channels are formed. These two layers are prepared according to the
general methods set forth earlier in this section. The resulting two
layer structure is then placed on top of the first layer onto which the
reagents have been deposited. A specific example of the composition of
the three layers is as follows (ration of component A to component B):
first layer (sample layer) 30:1 (by weight); second layer (flow channel
layer) 30:1; and third layer (control layer) 4:1. It is anticipated,
however, that other compositions and ratios of the elastomeric components
can be utilized as well. During this process, the reaction sites are
aligned with the deposited reagents such that the reagents are positioned
within the appropriate reaction site.

C. Partitioning, Detection and Analysis of Nucleic Acids

[0048] In this section, methods for analysis of nucleic acids in a sample
are described. The methods involve massive partitioning of the sample and
any nucleic acid molecules it contains, and amplification (as defined
herein) of target sequences in the partitioned nucleic acid molecules.
Various versions of the methods may also involve application of
particular amplication strategies, pooling of amplification products,
analysis of pooled amplification products and/or other features that will
be apparent upon reading this disclosure. This section also describes
devices (i.e., massively partitioning devices, MPDs) useful in carrying
out analyses according to the method.

[0049] Analytical methods described in this section can be used for
deleting the presence or absence of a target sequence, detection of
polymorphisms; single polynucleotide polymorphism (SNP) analysis;
haplotype analysis; amplification of a segment for sequence
determination, gene expression analysis, quantification of nucleic acids,
analysis of cells (see Section D, below), as well as other applications
that will be apparent to one of skill guided by this disclosure. Although
this section focuses on analysis of nucleic acids it will be appreciated
by the reader that many aspects of the description in this section will
be applicable, with appropriate modification, to analysis of other
molecules and of cells. FIGS. 3A and 3B are flow charts illustrating
partition and analysis of nucleic acids in which multiple targets are
amplified in which amplicons may be pooled. FIG. 3B illustrates partition
and analysis of nucleic acids in which, in one embodiment, target
molecules in only a single chamber are amplified.

[0050] i) Samples Containing Nucleic Acids

[0051] In one step of the assay method, a sample containing a plurality of
nucleic acid molecules is partitioned into a plurality of sub-samples, at
least two of which each comprise at least one nucleic acid molecule.

[0052] Samples that may be analyzed according to the invention are any
fluid sample that contains nucleic acids. A variety of types of samples
can be used, so long as at least some nucleic acids can be partitioned
from each other by the MPD. The nucleic acids can be free in solution or
can be contained in particles or within cells suspended in a fluid.
Samples may be processed so that any nucleic acids in the sample can be
amplified. For example, in samples containing cells or viruses, the cells
or viruses can be lysed or disrupted, using such routine methods as
exposure to enzymes (such as lysozyme), detergents, denaturants (such as
guanidine salts) and/or physical disruption) before the sample. Any
method of liberating nucleic acids that results in nucleic acid molecules
sufficiently intact and purified to amplify fragments is suitable.
Examples of samples containing nucleic acids are cell lysates or cell
fractions, water samples containing microorganisms, purified DNA
resuspended in an aqueous buffer, sputum, blood, nucleated blood cells,
tissue or fine needle biopsy samples, urine, peritoneal fluid, fecal
samples, and fleural fluid, or cells therefrom. Exemplary samples include
cells and cell lysates (e.g., eukaryotic cells, human cells, animal
cells, plant cells, fetal cells, embryonic cells, stem cells, blood
cells, lymphocytes, bacterial cells, recombinant cells and cells infected
with a pathogen, tissue samples), viruses, purified or partially purified
DNA or RNA, environmental samples (e.g., water samples), food samples,
forensic samples, plant samples and the like. It will apparent that the
sample can contain other compounds and macromolecules in addition to
nucleic acids. If necessary for the functioning of a microfluidic device
non-nucleic acid components and/or particulates can be removed by
filtration, sedimentation or other methods.

[0053] In one embodiment of the invention, the nucleic acids are contained
in cells, organelles, or viruses and the nucleic acids are not released
(e.g., the cells are not lysed) until at least after a partitioning step.
Particular aspects of this embodiment are discussed in Section D, below.

[0054] Analysis of nucleic acids in a sample generally involves
determining whether the sample contains a nucleic acid having a
particular target sequence. A target sequence may be predefined (i.e.,
known prior to analysis) or may be a sequence in a segment of a nucleic
acid defined by other parameters (e.g., defined as the segment of a gene
that can be amplified by a particular primer pair).

[0055] A target sequence can be any nucleic acid sequence of interest,
such as a sequence associated with a gene, a sequence that identifies a
particular allele or polymorphism, a sequence that, alone in combination
with other genotypic or phenotypic markers, identifies the presence in
the sample of a particular organism or strain, and the like. A target
sequence can also include sequences flanking a sequence of interest, such
as the sequences flanking a SNP. In addition, in some cases as will be
recognized from context, a "target sequence" is a sequence added during
an amplification step. For example, the B and U sequences discussed below
in the context of a Universal Amplification method can be referred to as
"target sequences" recognized by a probe or amplification primer.

[0056] A target sequence can be found in DNA (including genomic,
mitochondrial DNA, viral DNA, recombinant DNA and complementary cDNA made
from RNA) or in RNA (including rRNA, mRNA and iRNA). If a target sequence
is detected in a sample it is possible to deduce that the sample contains
a nucleic acid molecule containing the detected sequence or its
complement. For example, a sample from a human patient can be analyzed to
determine whether a viral nucleotide sequence (the target sequence) is
detectable in the sample, in order to diagnose (or rule out) viral
infection. As another example, genomic DNA from a human patient can be
analyzed to determine whether a particular polymorphism is or is not
present in a subject's genome.

[0057] In some cases, it will be advantageous to fragment the nucleic acid
molecules prior to the partitioning step. For example, to characterize
two genes on different regions of a eukaryotic chromosome it may be
useful to fragment the DNA to produce smaller nucleic acid molecules so
that the genes can be separately partitioned (i.e., partitioned into
different sub-samples). Fragmentation can be accomplished enzymatically
(e.g., using restriction enzymes), mechanically or chemically. In one
embodiment, shearing is accomplished by passing the DNA through a channel
of a MPD with a diameter (bore size) that is sufficiently small, or which
varies in diameter along the length of the channel, so as to sheer large
nucleic acids as they pass through. A sample containing a single DNA
molecule (e.g., a single chromosome) contains a plurality of nucleic
acids upon fragmentation of the single molecule.

[0058] ii) Partitioning of a Sample Containing Nucleic Acid Molecules

[0059] Methods for partitioning a sample using a MPD are provided in
Section B, above. The terms "to partition," "partitioning,"
"partitioned," and grammatical equivalents refer to the process of
separating a sample into a plurality of sub-samples using a MPD. A sample
is partitioned by introducing the sample into the flow channels and
reaction sites with valves open and then closing the valves to isolate
each sub-sample. It will be recognized that each sub-sample is contained
(for at least a period of time) in a separate reaction chamber such that
the sample is isolated from (not in fluidic communication with) other
sub-samples. It is sometimes convenient to refer to the "sample" even
after it has been partitioned into subsamples. Thus, in some contexts
"sample" can refer to the aggregate contents of the subsamples or
chambers after partition, as well as before partition.

[0060] When a sample containing a complex mixture of nucleic acid
molecules it is partitioned into very small-volume sub-samples, the
effective concentration of the target sequence in the sub-sample(s) in
which it is located is significantly increased. Effective concentration
of the target occurs because, while the number of molecules of target in
the sample does not change as a result of the partitioning, the number of
other molecules (including molecules that can produce side reactions,
e.g., primer-dimers and non-complementary DNA sequences in the sample) is
linearly proportional to volume. For example, if a 30 microliter sample
containing one molecule of interest is partitioned into ten thousand
subsamples (each with a volume of 3 nanoliters) the effective
concentration of molecule of interest is enriched by a factor of 104
in the chamber in which it is located. Since the ratio of target to side
reactions is inversely proportional to volume, partitioning into a small
volume increases this ratio (i.e., effectively concentrates). As noted by
McBride et al., such an increase in effective concentration results in
remarkable sensitivity and fidelity of PCR-based detection.

[0061] Typically the sample is partitioned into at least about 103
different subsamples or reaction chambers, sometimes at least about
5×103 different sub-samples or chambers, sometimes 104
different sub-samples or chambers, often at least about 2×104
different sub-samples or chambers, sometimes at least about
3×104 different sub-samples or chambers, and sometimes at
least about 105 different sub-samples or chambers. In certain
embodiments the sample is partitioned into between 100 and 100,000
sub-samples, more often between 1000 and 50,000 sub-samples, and
sometimes between 1000 and 20,000 sub-samples.

[0062] Typically the volume of each sub-sample is less than about 1000
picoliters (pL), often less than about 500 pL, sometimes less than about
100 pL, and sometimes less than about 50 pL.

[0063] The relationship between the number of nucleic acid molecules (or
non-nucleic acid macromolecules, particles or cells) in a sample, the
number of chambers into which the sample is partitioned, and the
distribution of number of nucleic acid molecules or other entities in
each chamber can be estimated using well know methods. For example, to
determine the number of chambers (C) into which the number (N) particles
(e.g. cells, nucleic acid molecules, etc.) would be partitioned so that
most or essentially all of the chambers contained either 0 or 1 particle
can be determined using a Poisson Distribution:

P ( x , λ ) = - λ λ x x !
. [ 1 ] ##EQU00001##

P(x,λ) is the probability of finding x particles if the average
number of particles in a box is λ. We can get this by setting the
average number (λ) such that

The average number of particles in a box is λ=N/C, so if you know
λ and N, you can find C:

C = N λ ##EQU00003##

λ is easily determined using eqn. [1] above. For instance, if x is
0.99 (i.e. 99% chance of a chamber containing a 0 or 1 particle),

e.sup.-λ(1--λ)≧0.99

λ≦˜0.1487

If N is 10,000, then

C ≈ 10000 0.1487 ##EQU00004##

[0064] C 67250 chambers are required to have 99% likelihood of 0 or 1
particles per chamber. Although this calculation is provided for
illustration it will be understood that any method (emperical or
analytical) may be used. In some applications it will be useful to use
such a calculation and adjust (e.g., dilute) the sample and/or select a
MPD with an appropriate number of chambers for an increased likelihood
there will be few, if any, chambers with more than a predetermined number
of target molecules (e.g., 1) per chamber.

[0065] iii) Amplification of Partitioned Nucleic Acids

[0066] Following the partitioning step, any target sequences of interest
that are in the sample are amplified. As used herein, nucleic acid
"amplification" is a process that produces multiple nucleic acid
molecules (called "amplicons") based on the presence of a particular
target sequence. Most often the amplicons include a base sequence that is
the same as, or complementary to, the target sequence so that
amplification means that the number of copies of the target sequence
increases. These identical or complementary amplicons are the products,
for example and without limitation, of the Polymerase Chain Reaction
(PCR) [see, Dieffenbach and Dvksler, 1995, PCR Primer: A Laboratory
Manual. CSHL press, Cold Spring Harbor, USA]; Nucleic Acid Sequence Based
Amplification (NASBA) [see Sooknanan and Malek, 1995, BioTechnology
13:563-65] SPIA® Isothermal Linear Amplification, Ribo-SPIA,
X-SPIA® [Nugen Technologies, San Carlos Calif., see U.S. Pat. No.
6,251,639, WO 02/72772; US2003/0017591 A1]; the Ligase Chain Reaction
(LCR) [Wu and Wallace, 1989, Genomics 4:560; Landegren et al., 1988,
Science 241:1077]; Transcription amplification [Kwoh et al., 1989, Proc.
Natl. Acad. Sci. USA 86:1173]; Self-sustained sequence replication
[Guatelli et al., 1990, Proc. Nat. Acad. Sci. USA 87:1874]. In some
embodiments, however, an amplicon is a nucleic acid with a sequence
different from the target sequence and the process of amplification
consists of increasing the number of amplicons if the target is present
in a chamber, but not in the absence of the target sequence.

[0067] As noted above, typically the invention is used to simultaneously
assay for multiple different target sequences in the same sample (e.g.,
sequences of multiple different genes or gene segments, or alternative
sequences of a single gene). For example, provided with a patient blood
sample, it may be useful to assay the sample for the presence of several
(e.g., 10, 20 or 100) different sequences each characteristic of a
different pathogen. In one embodiment, the different sequences are
amplified in different reaction chambers. For example, an assay to detect
the presence of multiple mutations in different genes from an individual
sample will result in analysis of products in different reaction chambers
if the genes are on fragments distributed to different sub-samples. In
other embodiments, multiple different target sequences are assayed for
and/or detected in a single reaction chamber, such as, for example, when
the reaction chamber contains a single rare cell and the assay is
designed to analyse several genetic loci in the cell genome. For example,
an assay to determine which of several possible polymorphisms (e.g.
defining a haplotype) are represented at a specific genomic site,
multiple primers or probes may be used to determine which of several
possible target sequences are present in a single reaction chamber. Very
often both types of amplification are used.

[0068] A variety of methods are known for "multiplex" analysis
(amplification of multiple sequences from one reaction) and can be
adapted for use in a single reaction chamber MPDs. In one embodiment
amplification is conducted using a universal primer strategy in which
each target sequence is initially amplified by a pair of target sequence
specific primers that include a 3-prime domain with a gene-specific
sequence and a 5-prime domain with a universal (not target specific)
sequence. For example, N different target sequences can be detected using
primers 5'-U1-FN-3' and 5'-U2-RN-3' where FN and
RN are forward and reverse PCR primers for each gene N, and U1
and U2 are sequences common to all of the primer pairs and Ui
and U2 may be the same or different. Subsequent rounds of
amplification can be conducted using primers 5'-U1-3' and
5'-U2-3'. See, e.g.,. Zhenwu Lin et al., 1996, "Multiplex genotype
determination at a large number of gene loci" Proc. Nat'l Acad. Sci USA
93: 2582-87. When used in an MPD this strategy allows use of a low
concentration of target specific primers (thereby reducing expense and
the chances of primer dimer and other unintended side reactions) and a
higher concentration of universal primers.

[0069] In one embodiment, the multiplex amplification is carried out in a
MPD using the primers and strategy described below in Section C(v).

[0070] Amplification reagents, including primers, can be provided by
prepositioning reagents in reaction chambers, by combining reagents with
the sample before partitioning, by a combination of prepositioning and
combining, or by any other suitable method. Specific amplification
reagents will depend on the amplification method and sample, but can
include primers, polymerase, reverse transcriptase, nucleotides,
cofactors, metal ions, buffers, and the like. Methods for prepositioning
reagents in a microfluidic device have been described (see McBride et
al., supra). In addition, detection reagents such as labeled probes can
be provided by prepositioning and/or combining.

[0071] To produce amplicons the environment of the MPD reaction chambers
is manipulated as required to accomplish amplification by the
amplification method selected. For example, thermocycling necessary for a
PCR-type amplification reaction can be accomplished by placing the device
on a thermocycling plate and cycling the device between the various
required temperatures for melting of the DNA duplex (either target or
amplicon), annealing of primers, and DNA synthesis. For example a
protocol with an initial ramp to 95° C. and maintain for 1 m;
three step thermocycling for 40 cycles [92° C. for 30 s,
54° C. for 30 s, and 72° C. for 1 m] or two step
thermocycling for 40 cycles [92° C. for 30 s and 60° C. for
60 s] can be used. A variety of thermocycling plates are available from
commercial sources, including for example the ThermoHybaid Px2 (Franklin,
Mass.), MJ Research PTC-200 (South San Francisco, Calif.), Eppendorf
Part# E5331 (Westbury, N.Y.), Techne Part# 205330 (Princeton, N.J.).

[0072] Although in some cases a single target sequence in the sample is
amplified, more often at least 2, at least 3, at least 5, at least 10, at
least 20, at least 30, at least 40, at least 50, or at least 100
different target sequences are amplified. Thus, in some embodiments,
sufficient reagents are provided to amplify more than one target
sequence.

[0073] Thus, the amplification procedure can produce from zero (if no
target sequence is present) to 100 or more different amplicons. Any
particular chamber may have zero, one or more than one amplicons species
(where amplicons corresponding to the same target sequence are of the
same "species") depending on the nature of the assay and sample.

[0074] Usually, at least about 103, at least about 104, at least
about 105, at least about 106, at least about 107, or at
least about 108 amplicon molecules are produced corresponding to
some or all of the target sequences present in the sample. Most
typically, from about 107 to about 109 amplicon molecules are
produced for each target sequence, although the number may be lower when
multiple reactions are conducted in a single reaction chamber.

[0075] iv) Detection of Amplicons

[0076] The amplicon products can be detected in individual chambers and/or
they can be pooled for subsequent detection and analysis (as described
below in Section C(vi)). That is, amplicons can be detected and then
pooled, pooled without previously being detected, or detected and not
subsequently pooled.

[0077] Amplicons can be detected and distinguished (whether isolated in a
reaction chamber or at any subsequent time) using routine methods for
detecting nucleic acids. Amplicons comprising double-stranded DNA can be
detected using intercalation dyes such as SYBR®, Pico Green (Molecular
Probes, Inc., Eugene, Oreg.), ethidium bromide and the like (see Zhu et
al., 1994, Anal. Chem. 66:1941-48) and/or gel electrophoresis. More
often, sequence-specific detection methods are used (i.e., amplicons are
detected based on their nucleotide sequence). Examples of detection
methods include hybridization to arrays of immobilized oligo or
polynucleotides, and use of differentially labeled molecular beacons or
other "fluorescence resonance energy transfer" (FRET)-based detection
systems. FRET-based detection is a preferred method for detection. In
FRET-based assays a change in fluorescence from a donor (reporter) and/or
acceptor (quencher) fluorophore in a donor/acceptor fluorophore pair is
detected. The donor and acceptor fluorophore pair are selected such that
the emission spectrum of the donor overlaps the excitation spectrum of
the acceptor. Thus, when the pair of fluorophores are brought within
sufficiently close proximity to one another, energy transfer from the
donor to the acceptor can occur and can be detected. A variety of assays
are known including, for example and not limitation, template extension
reactions, quantitative RT-PCR, Molecular Beacons, and Invader assays,
these are described briefly below.

[0078] FRET and template extension reactions utilize a primer labeled with
one member of a donor/acceptor pair and a nucleotide labeled with the
other member of the donor/acceptor pair. Prior to incorporation of the
labeled nucleotide into the primer during an template-dependent extension
reaction, the donor and acceptor are spaced far enough apart that energy
transfer cannot occur. However, if the labeled nucleotide is incorporated
into the primer and the spacing is sufficiently close, then energy
transfer occurs and can be detected. These methods are particularly
useful in conducting single base pair extension reactions in the
detection of single nucleotide polymorphisms and are described in U.S.
Pat. No. 5,945,283 and PCT Publication WO 97/22719.

[0079] Quantitative Real Time PCR. A variety of so-called "real time
amplification" methods or "real time quantitative PCR" methods can also
be used to determine the quantity of a target nucleic acid present in a
sample by measuring the amount of amplification product formed during or
after the amplification process itself. Fluorogenic nuclease assays are
one specific example of a real time quantitation method which can be used
successfully with the devices described herein. This method of monitoring
the formation of amplification product involves the continuous
measurement of PCR product accumulation using a dual-labeled fluorogenic
oligonucleotide probe--an approach frequently referred to in the
literature as the "TaqMan" method. See U.S. Pat. No. 5,723,591

[0080] Molecular Beacons: With molecular beacons, a change in conformation
of the probe as it hybridizes to a complementary region of the amplified
product results in the formation of a detectable signal. The probe itself
includes two sections: one section at the 5' end and the other section at
the 3' end. These sections flank the section of the probe that anneals to
the probe binding site and are complementary to one another. One end
section is typically attached to a reporter dye and the other end section
is usually attached to a quencher dye. In solution, the two end sections
can hybridize with each other to form a hairpin loop. In this
conformation, the reporter and quencher dye are in sufficiently close
proximity that fluorescence from the reporter dye is effectively quenched
by the quencher dye. Hybridized probe, in contrast, results in a
linearized conformation in which the extent of quenching is decreased.
Thus, by monitoring emission changes for the two dyes, it is possible to
indirectly monitor the formation of amplification product. Probes of this
type and methods of their use are described further, for example, by
Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer, 1996,
Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat. Biotechnol.
16:49-53 (1998).

[0081] Scorpion: The Scorpion detection method is described, for example,
by Thelwell et al. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas
et al., 2001, "Duplex Scorpion primers in SNP analysis and FRET
applications" Nucleic Acids Research 29:20. Scorpion primers are
fluorogenic PCR primers with a probe element attached at the 5'-end via a
PCR stopper. They are used in real-time amplicon-specific detection of
PCR products in homogeneous solution. Two different formats are possible,
the `stem-loop` format and the `duplex` format. In both cases the probing
mechanism is intramolecular. The basic elements of Scorpions in all
formats are: (i) a PCR primer; (ii) a PCR stopper to prevent PCR
read-through of the probe element; (iii) a specific probe sequence; and
(iv) a fluorescence detection system containing at least one fluorophore
and quencher. After PCR extension of the Scorpion primer, the resultant
amplicon contains a sequence that is complementary to the probe, which is
rendered single-stranded during the denaturation stage of each PCR cycle.
On cooling, the probe is free to bind to this complementary sequence,
producing an increase in fluorescence, as the quencher is no longer in
the vicinity of the fluorophore. The PCR stopper prevents undesirable
read-through of the probe by Taq DNA polymerase.

[0082] Invader: Invader assays (Third Wave Technologies, Madison, Wis.)
are used particularly for SNP genotyping and utilize an oligonucleotide,
designated the signal probe, that is complementary to the target nucleic
acid (DNA or RNA) or polymorphism site. A second oligonucleotide,
designated the Invader Oligo, contains the same 5' nucleotide sequence,
but the 3' nucleotide sequence contains a nucleotide polymorphism. The
Invader Oligo interferes with the binding of the signal probe to the
target nucleic acid such that the 5' end of the signal probe forms a
"flap" at the nucleotide containing the polymorphism. This complex is
recognized by a structure specific endonuclease, called the Cleavase
enzyme. Cleavase cleaves the 5' flap of the nucleotides. The released
flap binds with a third probe bearing FRET labels, thereby forming
another duplex structure recognized by the Cleavase enzyme. This time the
Cleavase enzyme cleaves a fluorophore away from a quencher and produces a
fluorescent signal. For SNP genotyping, the signal probe will be designed
to hybridize with either the reference (wild type) allele or the variant
(mutant) allele. Unlike PCR, there is a linear amplification of signal
with no amplification of the nucleic acid. Further details sufficient to
guide one of ordinary skill in the art are provided by, for example,
Neri, B. P., et al., Advances in Nucleic Acid and Protein Analysis
3826:117-125, 2000) and U.S. Pat. No. 6,706,471.

[0083] Padlock probes: Padlock probes (PLPs) are long (e.g., about 100
bases) linear oligonucleotides. The sequences at the 3' and 5' ends of
the probe are complementary to adjacent sequences in the target nucleic
acid. In the central, noncomplementary region of the PLP there is a "tag"
sequence that can be used to identify the specific PLP. The tag sequence
is flanked by universal priming sites, which allow PCR amplification of
the tag. Upon hybridization to the target, the two ends of the PLP
oligonucleotide are brought into close proximity and can be joined by
enzymatic ligation. The resulting product is a circular probe molecule
catenated to the target DNA strand. Any unligated probes (i.e., probes
that did not hybridize to a target) are removed by the action of an
exonuclease (which may be introduced before or after a pooling step).
Hybridization and ligation of a PLP requires that both end segments
recognize the target sequence. In this manner, PLPs provide extremely
specific target recognition.

[0084] Using universal primers, the tag regions of circularized PLPs can
be amplified and resulting amplicons detected. For example, TaqMan real
time PCR can be carried out to detect and quantitate the amplicon. The
presence and amount of amplicon can be correlated with the presence and
quantity of target sequence in the sample. For descriptions of PLPs see,
e.g., Landegren et al., 2003, Padlock and proximity probes for in situ
and array-based analyses: tools for the post-genomic era, Comparative and
Functional Genomics 4:525-30; Nilsson et al., 2006, Analyzing genes using
closing and replicating circles Trends Biotechnol. 24:83-8; Nilsson et
al., 1994, Padlock probes: circularizing oligonucleotides for localized
DNA detection, Science 265:2085-8.

[0086] As described above, a variety of multiplex amplification systems
can be used in conjunction with the present invention. In one type,
several different targets can be detected simultaneously by using
multiple differently labeled probes each of which is designed to
hybridize only to a particular target. Since each probe has a different
label, binding to each target to be detected based on the fluorescence
signals. By judicious choice of the different labels that are utilized,
analyses can be conducted in which the different labels are excited
and/or detected at different wavelengths in a single reaction. See, e.g.,
Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York,
(1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel
Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of
Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths,
Colour and Constitution of Organic Molecules, Academic Press, New York,
(1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and
Haugland, Handbook of Fluorescent Probes and Research Chemicals,
Molecular Probes, Eugene (1992).

[0087] Conventional multiplex fluorescence detection using many different
probes is limited, however, by fluorescence background because probe
concentration must be high enough to allow detection of many different
probes (i.e., one for each sequence to be detected). Combining many
probes results in fluorescence that is the sum of all the probes. This
also results in a fluorescence background that is the sum of the
background from all of the probes. This background may be so high as to
interfere with detection of the reaction product(s). An alternative
approach, referred to as the Universal Amplification ("UA") method uses
multiple sets of primer pairs, referred to here as "UA primer"
amplification in a PCR-type reaction. Universal amplification allows many
different sequences in a sample to be amplified using a single reaction
mixture, with lower background and cost than conventional systems, and is
particularly well suited for use with an MPD.

[0088] UA primers can be used to determine whether or not any one or more
of a number of different target nucleic acid sequences are present in a
sample (without necessarily identifying which of the several target
sequences is present). If the assay indicates that at least one of the
different nucleic acid sequences is present in the sample, subsequent
analysis can be conducted to determine which of the different sequences
is present. Such two-step analysis is advantageous in many applications.
For example, using the present invention, in a first step a sample can be
assayed to determine whether any of 100 (for example) different
pathogenic agents is present in the sample. If it is determined in the
first step that at least one pathogenic agent is present, the sample can
be subjected to further analysis to identify and characterize the
particular pathogen.

[0089] In one aspect the invention provides a method for detecting
multiple amplification products (i.e., amplicons) using the Universal
Amplification method. The total number of target sequences detected is
usually at least two, and is sometimes at least 5, more often at least
10, at least 20 or at least 30. In some embodiments the total number of
target sequences is between 2 and 100, between 5 and 100, between 10 and
100, between 20 and 100, or between 30 and 100. In some embodiments the
total number of target sequences is between 2 and 50, between 5 and 50,
between 10 and 50, between 20 and 50, or between 30 and 50. In some
embodiments the total number of target sequences is more than 100. The UA
method makers use of three or more types of primers:

[0090] The Type 1 primer has the structure 5'-UF-BXFN-3'
where UF is a universal forward primer sequence, Bx is a
sequence recognized by a detectable (e.g., detectably labeled) labeled
probe, Probe X, and F is a forward primer sequence specific to a target
sequence N so that F1 is primer for a first target sequence, F2
is a primer sequence for a second target sequence, and so on. Probe X can
be a molecular beacon, Taqman-type probe, or other probe (such as, but
not limited to, those described above) that specifically binds or
hybridizes to sequence B.

[0091] The Type 2 primer has the structure 5'-UR-RN-3' where
UR is a universal reverse primer sequence and RN is a primer
specific to a target sequence N so that R1 is primer for a first
target sequence, R2 is a primer for a second target sequence and so
on. UR may or may not be the same sequence as UF. In one
embodiment, 5'-UR-3' has the same sequence as 5'UF-3'.

[0094] Each pair of Type 1 and Type 2 primers is specific to a particular
target. The cognate pair of primers that amplify the same target is a "UA
primer pair." Thus, if there are 20 target sequences to be detected (for
example, sequences corresponding to 20 different pathogens) twenty
different primer pairs can be prepared, i.e.,
5'-U1-Bx-F.sub.[1→20]-3' and
5'-U2'-R.sub.[1→20]-3'. The various pairs of Type 1 and Type
2 primers are combined at low concentrations with the sample, and Type 3
and Type 4 primers are added at a higher concentration. During the
initial rounds of amplification, the Type 1 and Type 2 primers will
amplify any target sequences present in a sample or sub-sample. It will
be appreciated that U1 and U2 sequences can be designed with
sequences not present (or unlikely to be present) in the initial sample
nucleic acid, to avoid amplification of non-target sequences in the
sample. For example, for analysis of human DNA, U1 and U2 can
be selected to have sequences not found on the human genome. During
subsequent rounds of amplification, the amplification products generated
in the first rounds of amplification are themselves amplified by the Type
3 and Type 4 primers. The resulting double stranded amplification
products will have the structure (showing one strand):

5'-U1-Bx-F1-target
sequence1-R1'-U2'-3'

5'-U1-Bx-F2-target
sequence2-R2'-U2'-3'

5'-U1-BxF3-target
sequence3-R3'-U2'-3'

5'-U1-Bx-F4-target
sequence4-R4'-U2'-3'

5'-U1-Bx-F5-target
sequence5-R5'-U2'-3'

5'-U1-Bx-F6-target
sequence6-R6'-U2∝-3'

etc.

where U1' is the complement of U1 and U2' is the
complement of U2. Each of the amplification products shown above can
be detected by a probe (e.g., molecular beacon, Invader probe, Scorpion
probe) that hybridizes to Bx. Thus, using the UA universal probes
described herein, a multiplicity of target sequences can be detected
using a single probe. Most of the amplification steps involve
amplification using a single primer (if U1 and U2 are the same)
or primer pair. Methods for designing probes that recognize a specified
sequence (e.g., Bx are well known. For example, Molecular Beacons
can be designed as described in Marras et al., 2003, Genotyping single
nucleotide polymorphisms with molecular beacons. In Kwok, P. Y. (ed.),
Single nucleotide polymorphisms: methods and protocols. The Humana Press
Inc., Totowa, N.J., Vol. 212, pp. 111-128. Molecular Beacons can also be
designed with the help of a dedicated software package called `Beacon
Designer,` which is available from Premier Biosoft International
(www.premierbiosoft.com). However, it will be appreciated that the
sequence of Bx can be, and generally is, an artificial sequence
(i.e., not found in the in the initial sample nucleic acid) that can be
recognized by the probe.

[0095] Primer concentration(s) will vary with the length, composition, and
nature of the sample and targets. Those primer pairs with sequences
specific to each of the targets (e.g., Type 1 and 2 primers) are required
only in the first few rounds of amplification and can be provided in very
small quantities (for example and not limitation, e.g., typically less
than about 50 nM, more often less than about 30 nM and sometimes less
than about 20 nM). Type 3 and 4 primers can be provided at somewhat
higher concentration (for example and not limitation, e.g., typically
from about 100 nM to 1 uM, such as from about 200 nM to about 900 nM).
The practicioner guided by this disclosure will be able to select
appropriate concentration using routine methods.

[0096] The method can be modified in a variety of ways to achieve
particular results. In one version of the method, a relatively small
number of probe sequences can be used with a larger number of unique
target sequences, with different classes of target specific sequences
associated with differently labeled probes. For example, if target
sequences 1-20 are characteristic of a viral pathogen, target sequences
21-40 are characteristic of a bacterial pathogen, and target sequences
41-45 are positive control sequences (human genes), amplification of a
human patient sample could give zero, one or more than one of the
following 45 amplification products:

[0100] By using differently labeled probes BV (hybridizes to Set 1
products), BB (hybridizes to Set 2 products), and BC
(hybridizes to Set 3 products) the classes of amplification products can
be detected and distinguished. Thus, if the human sample produced any
amplicon to which Probe BV hybridized and emitted signal it would
indicate that the patient was infected with one of 20 viruses. If
desired, the precise identity of the viral pathogen could be determined
in a second assay step. Similarly, if the human sample produced any
amplicon to which Probe BB hybridized and emitted signal (different
from the signal emitted by Probe BV) it would indicate that the
patient was infected with one of 20 bacteria. Other types of samples,
such as food or agricultural sample can be screened for many different
pathogens simultaneously and if any hits are detected the sample can be
selected for further analysis to determine which of the many pathogen(s)
was responsible for the signal. If there is no signal, the sample can be
concluded to be pathogen free.

[0102] Although the "Universal Amplification" method described above is
suited for use in MPD-based analyses, it will be appreciated that this
method can be used in a variety of formats (both microfluidic and
nonmicrofluidic).

[0103] vi) Pooling of Amplicons

[0104] In some embodiments of the invention, following amplification and
optional detection of amplicons, the contents of the sub-samples,
including any amplicons in them, are pooled (i.e., allowed to combine or
mix at least partially). Pooling combines the amplicons (if present) from
multiple sub-samples. Pooling can be accomplished by, for example,
opening the valves of an elastomeric microfluidic device in which
partitioning and amplification occurred such that the contents of
multiple sub-samples (e.g., at least about 103, at least about
5×103, at least about 104, at least about
2×104, at least about 3×104, or at least about
105 sub-samples) are in fluidic communication with each other,
constituting a "post-amplification sample" that consists of the contents
of all of the chambers. Amplicons can mix by diffusion, which can be
accelerated using thermal, mechanical, acoustic, or chemical energy. In
one embodiment, pooling occurs primarily as a result of active mixing
(e.g., by pumping the fluid through flow channels in the device using a
rotary peristaltic pump or other mechanism). Alternatively or in
combination with the methods above, a portion, all or substantially all
of the post-amplification sample can be pumped out of or otherwise
withdrawn from the device, thereby pooling and mixing any amplicons
present in the sample. Any method that results in a distribution of
amplicons sufficient to carry out subsequent detection steps may be used.

[0105] It is not necessary that the various amplicons diffuse (or are
mixed) to equilibrium in the post-amplification sample, but it is
desirable that sufficient mixing occur so that an aliquot of
post-amplification sample contains a number of molecules of each amplicon
(e.g., at least 10, at least 100, at least 1000, or at least 5000
molecules) from each sub-sample in which amplicons were produced. For
illustration, consider carrying out an amplification reaction that
produces 100,000 copies of each of three distinct amplicons, i.e.,
amplicon A in chamber 1, amplicon B in chamber 2, and amplicon C in
chamber 3. The sub-samples are pooled (by releasing valves) and one-fifth
of the volume of the post-amplification sample removed from the device or
from the partition region of the device (see Section C (viii), below). If
the amplicons had diffused to equilibrium in the post-amplification
sample, and assuming no loss of material, the one-fifth volume would
contain about 20,000 molecules of each of the three amplicons. If
diffusion was less than complete, the one-fifth volume could contain
unequal amounts of each amplicon, for example 50,000 molecules of
amplicon A, 20,000 molecules of amplicon B and 15,000 molecules of
amplicon C.

[0106] Alternatively, all or substantially all, of the post-amplification
sample (or substantially all of it) can be withdrawn from the device,
thereby mixing any amplicons present in the sample.

[0107] vii) Subsequent Analysis of Amplicons

[0108] Following pooling (e.g., diffusion and/or mixing) all or a portion
of the amplicon pool can be used for subsequent analyses. Typically the
amplicon pool is divided into a plurality of aliquots and each aliquot
separately analyzed to determine a property (e.g., a nucleotide sequence
or the presence or absence of a predetermined nucleotide sequence) of an
amplicon or amplicons in that aliquot. If desired, the volume of the
amplicon pool can be increased by addition of a suitable solution such as
aqueous buffer or a reaction mixture containing amplification and/or
detection reagents. The amplicon pool can be divided into aliquots
manually or can be divided using an appropriately designed MPD (see
Section C (viii), below).

[0109] It will be apparent from the discussion above that each of the
aliquots from the amplicon pool will contain essentially the same set of
amplicons (i.e., the same amplicon species will be represented in each
aliquot). Each of the aliquots can be used for a different analysis. For
example, a first aliquot can be assayed for the presence of a first
target sequence (e.g., a first SNP in an amplified gene sequence), a
second aliquot can be assayed for the presence of a second target
sequence (e.g., a second SNP in the same amplified gene sequence), a
third aliquot can be assayed for the presence of a third target sequence
(e.g., a first SNP of a different amplified gene) and so on. It will be
appreciated that target sequences of interest are not limited to SNPs.

[0110] The subsequent analysis of amplicons can be carried out using any
desired technique including, without limitation, hybridization to a
target nucleic acid or array of targets, PCR amplification, FRET-assays,
hybridization to probes, and the like.

[0111] viii) Devices

[0112] As noted above, massive partitioning is accomplished using an
elastomeric MPD. Subsequent analyses can also be accomplished using any
suitable assay. In certain embodiments, an elastomeric microfluidic
device is used for subsequent analyses. In some embodiments the initial
partitioning and amplification, the subsequent mixing of applications,
and the subsequent analysis of amplicons are carried out using different
sections (i.e., different banks) of the same the same device. For
example, an elastomeric device can be fabricated with three regions: a
first region that is a MPD in which target sequences, cells or molecules
are amplified in individual chambers to produce amplicons and then
allowed to mix to produce an amplicon pool, a second region (which can be
as small as a single flow channel) by which the amplicon pool is
transferred to the third region, and a third region having a plurality of
flow channels with a region of each flow channel defining a reaction site
in which subsequent analysis of the amplicon pool occurs. In one
embodiment, the flow channels in the third region are blind flow channels
with reaction sites near the channel terminus. A schematic of an
exemplary device is shown in FIG. 2. In this schematic five control
channels (1, 2, 3, 4 and 5) are shown in black. A branched flow channel
system is shown in gray. It will be appreciated, as discussed above, that
the flow channel configuration need not be branched. In the device shown,
a sample containing an amplification mixture and nucleic acids is
injected into inlet A with the values formed by control channels 1 and 2
open, and control channel 3 actuated (closed). Control channel 2 is then
actuated to isolate the sample in multiple chambers (B). The samples are
then subject to thermocycling and optionally detection of the
amplification products (e.g., using a commercially available fluorescence
reader). Control channel 1 is then closed and control channel 2 opened to
allow mixing of amplicons in the various chambers to produce an amplicon
pool, a portion of which is then pumped into blind channels (D) with
control channels 3, 4 (if present) and 5 open. Alternatively, control
channels 2 and 3 are both opened and a portion of the amplicon pool is
pumped to a mixing chamber (C) with control channel 4 closed. Control
channel 3 is then closed and channels 4 and 5 are opened and portion of
the amplicon pool is pumped into blind channels (D). Control channel 5 is
then closed isolating blind reaction chambers (E) and the subsequent
round of analysis takes place.

[0113] In one embodiment the device is configured so that reagents can be
added to the pooled amplicon sample; for example, the mixing chamber (C)
shown in FIG. 2 may be fluidically linked to a reservoir containing
reagents (e.g., nuclease, probes or primers) that can be added prior to
distributing the amplicon pool into a reaction chambers.

[0114] ix) Systems

[0115] In one aspect, the invention provides a system for analysis of
nucleic acids, proteins or cells comprising a massively partioning device
and an external collection reservoir for collection of an amplicon pool.
The external reservoir can be any type of container or tube that is
fluidically connected to the MPD, so that the contents of the MPD
chambers can be transferred from the MPD to the reservioir. Transfer can
be by displacement of the MPD contents using a displacement fluid, by
active pumping, or by other means.

[0116] In one aspect, the invention provides a system for analysis of
nucleic acids, proteins or cells comprising a massively partitioning
device, optionally including an integral or external reservoir for
collection of an amplicon pool, and an (additional) external component.
In one embodiment the MPD includes a region with a plurality of flow
channels defining a reaction sites for subsequent analysis of the
amplicon pool aliquots (e.g., the "third" region of the device described
above in Section (viii).

[0117] In one aspect, the invention provides a system for analysis of
nucleic acids, proteins or cells comprising a massively partitioning
device as described in Section (viii) above, where the MPD has three
regions: a first region that is a MPD in which target sequences, cells or
molecules are amplified in individual chambers to produce amplicons and
then allowed to mix to produce an amplicon pool, a second region (which
can be as small as a single flow channel) by which the amplicon pool is
transferred to the third region, and a third region having a plurality of
flow channels with a region of each flow channel defining a reaction site
in which subsequent analysis of the amplicon pool occurs.

[0119] In one aspect, the invention provides a system for analysis of
nucleic acids, proteins or cells comprising a massively partitioning
device, optionally including an integral or external reservoir for
collection of an amplicon pool, optionally including a plurality of flow
channels defining a reaction sites for subsequent analysis of the
amplicon pool aliquots, and optionally including and an additional
reagent positioned in the chambers of the MPD and/or the reaction sites
for subsequent analysis of the amplicon pool aliquots. Exemplary
additional reagents include enzymes (e.g., nuclease, polymerase, or
ligase); primers and probes (PCR primers, molecular beacons, padlock
probes, proximity ligation probes, Universal Amplification primers),
amplification reagents and the like.

[0120] For illustration and not limitation, particular systems may
comprise an MPD and a heat source (e.g., thermocycler) positioned to
regulate the temperature of the contents of reaction chambers. In one
embodiment, heat is transmitted from the heat source to the MPD by
conduction (e.g., the heat source being adjacent and in contact with the
MPD). In one embodiment the MPD is fixed (e.g., clamped) to the heat
source.

[0121] For illustration and not limitation, particular systems may
comprise an MPD and a signal detector positioned to detect signal
emanating from reaction chambers in the MPD system. In one embodiment, a
fluorescent signal is detected. In one embodiment the system includes an
appropriately programmed computer coupled to the signal detector capable
of storing information such as the position, intensity and/or duration of
a signal emanating from reaction chambers in the MPD system.

[0122] For example, when the MPD comprises reaction chambers for analysis
of amplicon pool aliquots, the signal detector, heat source, or other
component of the system may be associated with those reaction chambers,
reaction chambers produced in the partitioning step, or both. Other
particular systems may comprise a MPD comprising prepositioned reagents
in one or more reaction chambers or mixing chambers.

[0123] x) Illustrative Examples

[0124] The following prophetic examples are intended to illustrate aspects
of the invention. However, they are for illustration only and are not
intended to limit the invention in any fashion.

[0125] 1. SNP Analysis

[0126] In this illustration, 200 different genes of an individual are
screened for the presence of mutations.

[0127] A sample containing genomic DNA from the subject is obtained. A
small number of genome equivalents is sufficient for analysis. Thus the
sample may be from a small number of cells (for example, fewer than 10
cells, and as few as one cell) which may be treated to release and
fragment genomic DNA. Alternatively isolated or purified DNA may be used.
Usually the DNA is fragmented by shearing, enzymatic or chemical
cleavage, or other methods known in the art. In one embodiment the DNA is
sheared by transport through a channel with varying cross-dimensions.

[0128] The reagents for amplification of target sequences are added to the
sample. The amplification reagents include the following:

[0129] a) Primer pairs for each of the 200 gene segments to be analyzed.
The primer pairs can be selected, for example, to (i) amplify a target
polymorphic site sequence only if site has a particular sequence (i.e., a
specified SNP allele is present) or (ii) amplify the target polymorphic
site sequence using primers that flank the SNP site, so that the segment
is amplified without regard to what SNP is present. Each primer pair
includes both a target specific sequence and one of two 5' universal
sequences shared by all of the forward or all of the reverse primers,
allowing all of the amplicons produced using the target specific primers
to be amplified with the same universal primers.

[0130] b) A pair of universal primers capable of amplifying all of the
amplicons produced using the target specific primers.

[0131] Primers are selected so that all of the first round amplifications
(using target specific primers) can occur under the same reaction
conditions.

[0132] c) Amplification reagents (polymerase, cofactors, nucleotides,
metal ions, buffer, etc.). The reagents may be added to the sample before
partition, may be pre-positioned in the reaction chambers, or some
reagents may be added and others pre-positioned.

[0133] The sample is injected into the partitioning channel system of a
MPD having 40,000 chambers. After injection of the sample, valves are
closed creating 40,000 isolated reaction chambers. Each reaction chamber
contains all of the probes described above, and some of the chambers
contain a nucleic acid molecule with a target sequence of interest as a
consequence of the partitioning. The MPD is placed on a thermocycler and
cycled using an appropriate protocol (e.g., 2 min at 51° C., 1 sec
at 96° C. and 59 sec at 95° C., followed by 40 cycles of 1
min at 58° C., 1 sec at 96° C. and 59 sec at 95°
C.). Following amplification, the values are opened and sub-samples
allowed to mix (e.g., by diffusion or active mixing) producing the
amplicon pool.

[0134] A portion of the resulting amplicon pool is withdrawn from the MPD
and distributed into two hundred (200) aliquots, and each aliquot is
subjected to a different SNP assay. Alternatively, different sets of SNP
assays can be conducted using multiplex methods. The individual SNP
assays can be carried out using, for example, a Taqman®-type probe,
Molecular Beacon, Scorpion, or other detection methods and detected using
a fluorescence detector.

[0135] 2. Analysis of Many Sequences Using Nucleic Acids from a Single
Cell or Very Few Cells

[0136] In one embodiment, a nucleic acid analysis is conducted on a single
cell. Such an analysis is useful for diagnostic or prognostic methods
when tissue is limited such as, for example, genetic testing of a single
blastocyst of a pre-implantation embryo produced using in vitro
fertilization techniques. Such testing is also useful in the study or
cloning of non-human animals. For example, blastocyst cells obtained from
a non-human animal can be assayed for the presence, expression or
characteristics of a transgene or endogenous gene. It can be verified
that the genotype or expression profile of the embryo is consistent with
the goals of the researcher prior to implantation into a surrogate
mother, resulting in savings of time and resources. Such testing is also
useful in forensic analysis in which very few cells may be available or
in which cells must be analyzed individually because a sample is
contaminated with cells from multiple sources.

[0137] Analysis of nucleic acids of a single cell is illustrated by the
flow chart in FIG. 3c. It will be appreciated that the figure is provided
to assist the reader in understanding the invention, and is not intended
to limit the invention in any fashion.

[0138] In this method, the single cell (or small number of cells) is
provided in a solution or combined with a solution. The cell is treated
to release DNA. Any number of methods for cell lysis (e.g., using
sonication, denaturants, etc.) are suitable. If genomic DNA is being
analyzed it is fragmented. The desired fragment size will be based on the
method of detection of the target sequence and the number of reaction
chambers on the chip. The goal is to end up with large enough fragments
so that target sequences can be amplified (typically >300 bp) and
enough fragments so that different amplicons (i.e., amplicons
corresponding to different sequences) will be generated is separate
reaction chambers. Thus, the intended average size will depend on the
number of target sequences to be detected and the number of partitions
available. Fragments can be created by restriction digestion or other DNA
fragmentation methods. In one embodiment, DNA is sheared by driving it
thorough a narrow opening. Thus, a MPD can be designed with a via or flow
channel of sufficiently small diameter narrowness to shear DNA to the
desired fragment size. In an embodiment the diameter of the via or flow
channel varies across its length (e.g., narrow-wide-narrow-wide) to drive
the fragmentation. The sample is introduced into a MPD and the MPD valves
are actuated to partition the DNA fragments, or RNA molecules, into
separate reaction chambers.

[0139] Reagents sufficient to amplify each of the target sequences of
interest are provided in each reaction chamber. For purposes of this
example, assume 50 different loci containing SNPs are of interest, and at
each of the 50 polymorphic loci there are two different possible
sequences at the SNP site, with the 100 total different target sequences
designated SNP 1A, 1B, 2A, 2B, . . . 50A, and 50B. As discussed above,
the reagents can be added to the solution containing the intact cell, can
be prelocated in the reaction chambers, or some combination of the two.
In this example, the amplification reagents include primers sufficient to
amplify gene segments spanning each of the 50 loci to produce the SNP
site and 40 basepairs of flanking sequence on each side. UA amplification
primers may be used. Amplification is then carried out (e.g., by
thermocycling if the amplification method is PCR or reverse
transcription-PCR). Amplicons are produced in those reaction chambers
that contain a nucleic acid molecule (i.e., DNA fragment or RNA molecule)
comprising one of the target sequences.

[0140] As described above, following amplification the valves are opened
and amplicons allowed to mix to produce an amplicon pool. The pool is
than divided into 100 different aliquots (optionally using a duel bank
MPD as described in Section C (viii), above). In each aliquot a single
assay is carried out for an individual SNP using, for example, a
Taqman®-type probe, Molecular Beacon, Scorpion, or other detection
methods known in the art.

[0141] 3. Detection of Pathogens Using the UA System

[0142] In this illustration, a sample is assayed for the presence of 150
different pathogens. Exemplary samples for the method include (i) a
sample is obtained from a patient, (ii) an environmental sample (e.g.,
from a pond or reservoir) and (iii) a sample from a poultry processing
facility.

[0143] The reagents for amplification of target sequences are added to the
sample. The amplification reagents include the following:

[0144] a) Fifty UA primer pairs for gene segments found in fifty different
bacterial pathogens. The forward primer of these UA primer pairs includes
a recognition site for a molecular beacon labeled with the blue
fluorescing dye Cy 5.5.

[0145] b) Fifty UA primer pairs for gene segments found in fifty different
fungal pathogens. The forward primer of these UA primer pairs includes a
recognition site for a molecular beacon labeled with the green
fluorescing dye 6-FAM (Fluorescein).

[0146] c) Fifty UA primer pairs for gene segments found in fifty different
viral pathogens. The forward primer of these UA primer pairs includes a
recognition site for a molecular beacon labeled with the red fluorescing
dye Cy 3.

[0148] Primer sequences are selected so that all of the UA primer pairs
produce amplicons that can be amplified using the same Type 3 and Type 4
primers; and all of the first round amplifications can occur under the
same reaction conditions.

[0149] e) Molecular beacons that recognize the recognition sites of the
three UA forward primers and are labeled as indicated above.

[0150] f) Amplification reagents (polymerase, cofactors, nucleotides,
metal ions, buffer, etc.). The reagents may be added to the sample before
partition, may be prepositioned in the reaction chambers, or some
reagents may be added and others prepositioned.

[0151] The sample is injected into the partitioning channel system of a
MPD having 40,000 chambers. After injection of the sample each valves are
closed creating 40,000 isolated reaction chambers. Each reaction chamber
contains all of the probes described above, and some of the chambers
contain a nucleic acid molecule with a target sequence of interest. The
MPD is placed on a thermocycler and cycled using an appropriate protocol
(e.g., 2 min at 51° C., 1 sec at 96° C. and 59 sec at
95° C., followed by 40 cycles of 1 min at 58° C., 1 sec at
96° C. and 59 sec at 95° C.). Following amplification, the
device is imaged using a commercially available, modified, or custom made
fluorescence reader.

[0152] The appearance of chambers fluorescening blue indicates that there
is at least one bacterial pathogen present in the sample. The appearance
of chambers fluorescening green indicates that there is at least one
fungal pathogen present in the sample. The appearance of chambers
fluorescesing red indicates that there is at least one viral pathogen
present in the sample.

[0153] If none of the chambers is emits blue, green or red fluorescence
when illuminated at the proper wavelengths this is an indication that
none of the pathogens tested for are present (typically assays would also
include a positive control). If, however, red fluorescence was detected,
this would be an indication that a virus is present. Further testing
could then be carried out to identify the viral pathogen. For further
testing, the values of the MPD are opened and sub-samples allowed to pool
by diffusion or active mixing, producing the amplicon pool. A portion of
the resulting amplicon pool is withdrawn from the MPD and divided into
fifty (50) aliquots each containing reagents for identification of one of
the 50 viruses assayed for in the initial part of the screen. An
exemplary detection method uses 50 molecular beacons (one in each
aliquot) that each recognize a different virus specific sequence in the
amplicon. By determining which of the molecular beacons bind a target
sequence present in the amplicon pool (or the portion contained in an
aliquot) the identity of the pathogen is determined.

[0154] 4. Additional Applications

[0155] It will be appreciated to the reader that the methods of the
invention can be used in many applications not specifically described,
including, for example and not limitation, detection of gene mutations
(substitutions, deletions, translocations, amplifications, etc.) in
samples from cancer patients and others. Many of these assays can be
carried out without using the optional pooling step and subsequent
analysis steps.

D. Partitioning, Detection and Analysis of Proteins and Other Biomolecules

[0156] Proteins and other biomolecules can be partitioned in a manner
analogous to that described above for nucleic acid molecules. Any
suitable method can be used to produce an amplification product
indicative of the presence of a protein. In one method, a proximity
ligation procedure is used. The proximity ligation procedure is analogous
in certain respects to the use of padlock probes, described above in
Section C, but is used for detecting proteins and other analytes. The
proximity ligation procedure uses specific protein binding agents linked
to oligonucleotides. Examples of specific protein binding agents include,
but are not limited to, antibodies (defined as any specific binding agent
comprising a CDR, including phage display antibodies, single chain
antibodies, monoclonal antibodies, and the like) and nucleic acid
aptamers. The proximity ligation procedure is described in Landegren et
al., 2003, supra; Landegren et al., 2004, Molecular tools for a molecular
medicine: analyzing genes, transcripts and proteins using padlock and
proximity probes, J Mol Recognit. 7:194-7; Gullberg et al., 2004,
Cytokine detection by antibody-based proximity ligation, Proc Natl Acad
Sci USA 101(22):8420-4; Fredriksson et al., 2002, Protein detection using
proximity-dependent DNA ligation assays, Nat Biotechnol. 20:448-9; and
Landegren, 2002, Methods and kits for proximity probing United States
Patent Application 20020064779. Briefly, a pair of protein binding agents
that recognize different epitopes of a target protein are used. Each of
the binding agents is attached (e.g., via streptavidin-biotin linkage) to
a synthetic DNA strand that includes a PCR primer binding site. The
synthetic DNA strands are brought into proximity when both binding agents
bind the same target molecule. A connector oligonucleotide that
hybridizes to sequences at the ends of both of the synthetic DNAs is
added in excess, bringing termini of the DNA strands together so that
they can be joined by ligase. In the presence of PCR reagents and primers
that recognize the primer binding sites on the two DNA strands, a region
of the ligated sequence may be amplified and detected by real time PCR.
In contrast, unligated strands are not amplified and therefore not
detected in the assay.

[0157] It will be appreciated that the assay also may be used to assay for
non-protein molecules that are specifically bound by a nucleic acid
aptamer, antibody or other binding agent. Proximity ligation methods can
also be used to detect nucleic acid targets. In this approach, nucleotide
sequences complementary to the target are used rather than protein
binding agents.

E. Partitioning of Cells

[0158] In another aspect of the invention, individual cells are isolated
by partitioning using a MPD, and one or more properties of one or more of
the individual cells are determined. Using this method, analysis of
individual cells can be carried out without background from other cells
in a sample.

[0159] Virtually any property of an individual cell can be assayed. For
illustration, cell properties include

[0160] the presence or absence of a target nucleic acid sequence in the
cell (where the nucleic acid is RNA or DNA; recombinant or naturally
occurring; cellular or viral; nuclear, cytoplasmic or from an organelle);

[0161] the presence or absence of a protein or epitope in the cell or on
the cell surface;

[0162] secretion by the cell of a protein or non-protein molecule, for
example in response to a stimulus;

[0163] metabolic reactions or changes in cell metabolism, for example in
response to a stimulus;

[0164] other properties of cells (which will be recognized by those of
skill in the art);

[0165] combinations of two, three, or more than three different properties
(e.g., the presence in a cell of two different target nucleic acid
sequences; the presence in a cell of a target nucleic acid sequence and a
protein epitope; a change in a metabolic property of a cell and
expression of a nucleic acid sequence in the cell; a cell surface epitope
and secretion of a cytokine by the cell in response to a stimulus, etc.).

[0166] For this analysis, a liquid sample containing a plurality of
separable cells is introduced into the MPD and the cells partitioned. A
"separable cell" is a cell that is physically separated from other cells
and can be partitioned into a chamber without other cells. In some cases
(e.g., blood cells, lymphocytes, spermatocytes, oocytes, yeast, certain
bacteria or other microorganisms) seperable cells can be obtained from a
patient or other source with little processing. In other cases (e.g.,
liver biopsy, cultured cells, blastocyte) it will be necessary to disrupt
a tissue or aggregate mechanically, enzymatically, or using other methods
well known in the art. See, e.g., Ausubel et al., 2004, Current Protocols
In Molecular Biology, Greene Publishing and Wiley-Interscience, New York;
Chapter 25. Examples of cells that can be assayed in this method include
eukaryotic cells, human cells, animal cells, plant cells, fetal cells,
embryonic cells, stem cells, blood cells, lymphocytes, bacterial cells,
recombinant cells and cells infected with a pathogen. Further, although
this section describes analysis of cells, the reader will appreciate that
the same methods can be used for analysis of other biological entities,
such as viruses and organelles.

[0167] In one aspect, the method includes partitioning a sample comprising
a plurality of seperable cells into at least 103 separate reaction
chambers in an MPD, where after partitioning at least two chambers
comprise exactly one cell each. Often the sample is partitioned into at
least 104 separate reaction chambers, at least 2×104
separate reaction chambers or at least 3×104 separate reaction
chambers. The number of cells introduced into the MPD and/or the number
of chambers in the MPD are selected so that most or virtually all of the
chambers contain either no cells or a single cell. This can be determined
from the Poisson distribution (based on the number of chambers in the
device and number of cells injected into the device) or empirically
(e.g., by detecting the number of chambers that contain cells). Usually
at least 90% of the chambers contain zero or one cell, often at least 99%
of the chambers contain zero or one cell, and in some cases virtually all
of the chambers contain zero or one cell.

[0168] Each of the plurality of chambers contains the same reagents for
conducting the analysis. All or some of these reagents can added to the
sample or cells prior to injection into the device and/or can be
prepositioned in the chambers and/or provided in inactive form, as
described above. Because the reagents are constant, any
chamber-to-chamber differences in analytical results are due to the
presence of different cells (or no cell) and reflect differences in the
properties of the cells. By detecting different signals from different
chambers, a property or properties of cells in chambers can be determined
and compared. This method finds a variety of applications in which it is
informative to determine that a sample contains a cell having two or more
properties detectable in separate assays. This method also finds a
variety of applications in which the cell of interest is a rare cell in a
background of many other cells.

[0169] The nature or type of reagents used will depend on the type of
assay contemplated and specific properties to be detected. Generally the
properties that can be assayed can be divided into two groups: properties
determined based on the presence or absence of a nucleic acid target
sequence and properties determined based on something other than the
presence or absence of a nucleic acid target sequence. In many
applications both a nucleic acid analysis and detection of a different
type of property are carried out.

[0170] In embodiments in which the analysis of cell properties includes a
detecting a nucleic acid sequence (i.e., one, two or more target
sequences are detected for an isolated cell) reagents suitable for
nucleic acid analysis include those used for nucleic acid amplification
(including but not limited to the PCR, SPIA, Invader, and other
amplification methods described in this disclosure or known in the art)
and those used for detection (including but not limited to, FRET based
methods and other detection methods described in this disclosure or known
in the art). In one embodiment the UA amplification/detection methods
described in Section C (v) are used.

[0171] Methods are known in the art for assay of a multitude of cell
properties other than or in addition to the characteristics of nucleic
acids. For example, proximity ligation and FRET-based assays can be used
to detect the presence of proteins or epitopes in a cell; presence,
activation or change in enzymatic activities; intracellular organelle
function; pathogen (e.g., viral) infection; intracellular signaling;
protein-protein interactions; protein-DNA interactions; colocalization of
proteins cell cycle; metabolic reactions such as generation of reactive
oxygen species; mitochondrial membrane potential; apotosis; intracellular
organelle function; changes in representations of cell types in cell
populations; and subcellular localization of macromolecules.

[0174] Many other assay methods are known or can be developed. Reagents
appropriate for each reaction type will be provided in the sample and/or
prepositioned in the reaction chamber. Exemplary reagents include
antibodies, ligands, enzyme substrates, effectors and the like.

Exemplary Applications

[0175] The following prophetic examples are intended to illustrate aspects
of the invention. However, these examples are for illustration only and
are not intended to limit the invention in any fashion.

[0176] 1. Detection and Characterization of Pathogens

[0177] The methods of the invention may be used for detection,
identification and characterization of pathogens. There are many
situations in which a sample contains a heterogeneous mixture of
microorganisms (e.g., various bacterial species or strains, viruses,
fungi) for which rapid detection and identification would be
advantageous. For example, clinical (patient) samples often contain small
numbers of microorganisms (e.g., bacteria, fungi) or viruses. Rapid
characterization would permit earlier administration of appropriate
drugs, if necessary. Similarly, the ability to rapidly detect and
identify cellular and viral pathogens would be of value in the medical,
veterinary, and agricultural fields, as well as in response to actual or
suspected bioterrorism and for rapid detection water or food
contaminants. In many cases a relatively small number of cells are
available to work with, and, as noted, the cells are often available as a
heterogeneous mixture with other cells.

[0178] In one illustrative embodiment, the method is used to determine
whether a patient is infected with methicillin-resistance S. aureus. S.
aureus can be identified using a bacteria-specific probe (e.g. to a rRNA
gene). An methicillin resistant strain is distinguished from
non-resistant strains based on a characteristic genetic sequence such as
an open reading frame (gene or gene segment) or single polynucleotide
polymorphism. A sample containing bacteria cells is obtained from a
patient (e.g., a nose swab containing about 100 bacterial cells) and
diluted into a reaction mixture containing nucleic acid primers and other
reagents for amplification and detection of target sequences (e.g., PCR
reagents, molecular beacons, polymerase, nucleotides, agents that lyse
cells for nucleic acid release, etc.). Exemplary PCR primers are
described in Huletsky et al., 2004, "New real-time PCR assay for rapid
detection of methicillin-resistant Staphylococcus aureus directly from
specimens containing a mixture of staphylococci." J Clin Microbiol.
42:1875-84. One primer/probe set in the reaction mixture emits a red
fluorescence signal in the presence of a S. aureus target sequence found
in both resistant and non-resistant stains while the second primer/probe
set emits a green fluorescence signal only in the presence of a S. aureus
target sequence found in the resistant strain. The cells are injected
into a MPD and control channels actuated to create separate reaction
chambers (e.g., a sample containing about 100 bacterial cells is
partitioned into 2000 chambers) all or most of which contain zero or one
cell. The device is placed on the thermocycler or amplification is
otherwise initiated. Detection in a chamber of only a red signal
indicates the presence in the sample of non-resistant S. aureus;
detection in a chamber of both a red and green signal indicates the
presence in the sample of drug resistant S. aureus; detection or no
signal indicates no S. aureus bacteria are present in the sample.

[0179] 2. Quantitation of Cells in a Population Having Specific Properties

[0180] In one aspect, the method is used for quantization of cells in a
heterogeneous population having specific properties. For illustration, a
cell population (e.g., peripheral blood mononuclear cells (PBMC))
containing cytotoxic T lymphocytes (CTL) (effector cells) can be
partitioned and the ability of the cells to be stimulated by an antigen
tested. The antigen reagent can be prepositioned in chambers or combined
with cells immediately before partition. The proportion or type of cells
activated in the presence can be assayed using any of a variety of assays
for effector cell activation. For example, by performing in vitro
stimulation after limiting dilution of circulating CTLs with the gag
antigens of human immunodeficiency virus (HIV), the precursor population
of gag-specific CTL can be quantitated and/or characterized. See, e.g.,
Koup "Limiting dilution analysis of cytotoxic T lymphocytes to human
immunodeficiency virus gag antigens in infected persons: in vitro
quantitation of effector cell populations with p17 and p24 specificities"
J Exp Med. 1991 Dec. 1; 174(6):1593-600.

[0181] 3. Characterization of a Rare Cell in a Background of Other Cells

[0182] It is often advantageous to quantitate and/or characterize rare
cells in a background of other cells. For example, in cancer, individual
disseminated cancer cells may be found in blood. Further, biopsies may
recover only a few malignant cells in a background of normal cells. The
methods of the present invention allow the malignant cells to be
isolated, identified based on a property (e.g., antigen, mutation or
expression pattern) unique to the cancer cell, and then a different
property of the cell determined.

[0183] In another example, nucleated fetal red blood cells are found at
low levels in the blood of pregnant women and are a potential source of
information about the fetal genome including any sequences associated
with disease or propensity to disease. However, even enriched 10,000-fold
fetal cells may be less than 0.1% of a sample making analysis by
conventional methods difficult. Cells from a sample enriched for fetal
NRBCs can be partitioned using the methods disclosed herein. Chambers
containing fetal cells can be identified using a fetal-specific probe
(e.g., a probe specific for the Y chromosome; abundance of RNA encoding
fetal forms of hemoglobin) and assayed for several genetic
characteristics using a multiplex assay. Other examples of rare cells in
a background of different cells include, for example, a virally infected
cell in a background of uninfected cells, a cell expressing a gene in a
background of cells not expressing the gene; and the like.

[0184] In one embodiment, the MPD is used to partition a mixed population
of cells to detect a property characteristic of a rare cell type in the
population, i.e., cells comprising less than about 1%, more often less
than about 0.1%, and very often less than about 0.01% of the cells in the
population. There are many cases in which it advantageous to determine
the properties of a rare cell in a population of other cells. The methods
of the present invention enable analysis of a rare cell without
background or interference for other cells. In general, the method
involves partitioning cells and assaying individual cells for at least
two properties at least one of which identifies the rare cell.

[0185] 4. Expression Analysis of Individual Cells

[0186] In one embodiment the nucleic acid being analyzed is or includes
RNA, and the expression level of specified genes in an individual cell is
determined. Again using the example of a virus-infected cell, the
expression profile for several host genes in a single cell can be
correlated with the presence or absence of virus or with viral load. Gene
expression profiles can also be correlated with cell identity (e.g.,
different expression profiles for different cells in a sample containing
a heterogeneous mixture of cells) or cell response to a stimulus (e.g.,
the presence of a ligand that binds a cell receptor).

[0187] Because expression analysis typically involves a quantitative
analysis, detection is typically achieved using one of the quantitative
real time reverse transcriptase PCR methods described above. Thus, if a
TaqMan approach is utilized, the reagents that are introduced (or
previously spotted) in the reaction sites can include one or all of the
following: primer, labeled probe, nucleotides and polymerase. Another
approach is Ribo-SPIA (see above).

[0188] While the present invention has been described with reference to
the specific embodiments thereof, it should be understood by those
skilled in the art that various changes can be made and equivalents can
be substituted without departing from the scope of the invention. In
addition, many modifications can be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
achieve the benefits provided by the present invention without departing
from the scope of the present invention. All such modifications are
intended to be within the scope of the claims appended hereto.

[0189] All publications and patent documents cited herein are incorporated
herein by reference as if each such publication or document was
specifically and individually indicated to be incorporated herein by
reference. Citation of publications and patent documents is not intended
as an indication that any such document is pertinent prior art, nor does
it constitute any admission as to the contents or date of the same.